Application of ZCCHC2 in sicca syndrome diagnosis and systemic lupus erythematosus distinguishing

Abstract

The invention relates to the technical field of disease diagnosis, in particular to application of ZCCHC2 in sicca syndrome diagnosis and systemic lupus erythematosus distinguishing.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the priority of Chinese patent application No. 2023106936175 filed with the Chinese Patent Office on June 12, 2023, entitled "Use of ZCCHC2 in the diagnosis of Sjögren's syndrome and differentiation of systemic lupus erythematosus", the entire contents of which are incorporated into this application by application. Technical Field

[0003] The present application relates to the field of disease diagnosis technology, and specifically to the use of ZCCHC2 in diagnosing Sjögren's syndrome and differentiating systemic lupus erythematosus, as well as its use in preparing related diagnostic products. Background Art

[0004] Sjögren's syndrome (SS) is a chronic inflammatory autoimmune disease that mainly affects exocrine glands. It is also known as autoimmune exocrine gland epithelial cell inflammation or autoimmune exocrine disease. In addition to dry mouth and dry eyes due to damaged salivary glands and lacrimal glands, other exocrine glands and other organs outside the glands are involved, resulting in symptoms of multi-system damage. There are multiple autoantibodies and hyperimmunoglobulinemia in the serum. The disease is divided into primary and secondary types. Primary Sjögren's syndrome is a global disease, with a prevalence of 0.3% to 0.7% in the Chinese population and 3% to 4% in the elderly population. The disease is more common in women, with a male-to-female ratio of 1:9 to 20. The age of onset is mostly between 40 and 50 years old, and it is also seen in children.

[0005] As people's understanding of SS continues to deepen, its classification and diagnostic criteria are constantly being proposed and improved in order to achieve higher diagnostic accuracy. At present, the most widely used in scientific research and clinical practice are the AECG classification criteria formulated by the European and American Consensus Group (AECG) in 2002 and the European League Against Rheumatism (EULAR)-ACR classification criteria officially published in 2016. Regarding pSS in children, there is also a 1999 recommendation for the diagnostic criteria for pSS in children (1999). However, due to the high heterogeneity of clinical manifestations of patients, clinical diagnosis is difficult, and there are deficiencies in the diagnosis of Sjögren's syndrome in China, such as: 1) China does not have corresponding diagnostic guidelines: in practice, the diagnostic guidelines of European and American countries (mainly ACR and EULAR standards) are often used. However, it has been confirmed that the etiology of Sjögren's syndrome is complex and heterogeneous, and there are huge differences in the detection results of markers such as autoantibodies in different races and regions. Therefore, although the diagnostic criteria for Sjögren's syndrome have high specificity and sensitivity for the European and American population, their diagnostic efficacy in the Chinese population needs to be further verified. 2) Complex diagnostic process: The current clinical diagnosis method is complex, including clinical symptoms and multiple tests such as immunology. Sjögren's syndrome is diagnosed after scoring. Misdiagnosis may occur, and it may be misdiagnosed as systemic lupus erythematosus (SLE), rheumatoid arthritis, allergic purpura, chronic hepatitis, respiratory system diseases and blood system diseases.

[0006] Therefore, continuing to explore the potential therapeutic or diagnostic targets for Sjögren's syndrome, exploring the mechanism of target action, and promoting new drug development and clinical transformation will be of great significance to the treatment of Sjögren's syndrome.

[0007] In view of this, this application is filed. SUMMARY OF THE INVENTION

[0009] In order to solve the above technical problems, the present application screened the genes or proteins differentially expressed in peripheral blood mononuclear cells of Sjögren's syndrome patients and normal people, and found that ZCCHC2 can be used as an indication of Sjögren's syndrome. What is more exciting is that ZCCH2 can distinguish Sjögren's syndrome from systemic lupus erythematosus. Therefore, it is proposed that the ZCCHC2 gene or ZCCHC2 protein can be used as a biomarker for the diagnosis of Sjögren's syndrome, and can also be used as a biomarker for distinguishing Sjögren's syndrome from systemic lupus erythematosus, improve the misdiagnosis of Sjögren's syndrome, make up for the shortcomings of existing Sjögren's syndrome related indicators, and have significant clinical value.

[0010] Therefore, this application has at least the following purposes:

[0011] The purpose of this application is to seek a completely new use, product or method for diagnosing Sjögren's syndrome; in some aspects, it is used to diagnose Sjögren's syndrome, and in other aspects, it is used to differentiate between Sjögren's syndrome and systemic lupus erythematosus.

[0012] To achieve the above objectives, this application proposes the following specific technical solutions:

[0013] The present application first provides the use of ZCCHC2 expression level as a marker in diagnosing Sjögren's syndrome; or; the use of ZCCHC2 expression level as a marker in distinguishing Sjögren's syndrome from systemic lupus erythematosus.

[0014] The present application also proposes the use of a reagent for detecting the expression level of ZCCHC2 in a sample in the preparation of a diagnostic product, wherein the diagnostic product includes a product for diagnosing Sjögren's syndrome; or a product for distinguishing Sjögren's syndrome from systemic lupus erythematosus.

[0015] 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.

[0016] In some aspects, the ZCCHC2 expression level serves as an independent indicator for the diagnosis of Sjögren's syndrome.

[0017] In some aspects, the ZCCHC2 expression level serves as an independent indicator for distinguishing Sjögren's syndrome from systemic lupus erythematosus.

[0018] In some aspects, the expression level comprises a nucleic acid expression level or a protein expression level.

[0019] In some aspects, when the expression level is a nucleic acid expression level, the reagent is suitable for detecting the nucleic acid expression level by sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology.

[0020] In some aspects, when the expression level is a protein expression level, the reagent for detecting the protein expression level is suitable for detecting the protein expression level by sequencing technology, immunological technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology.

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

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

[0023] Furthermore, the nucleic acid expression level is obtained or detected by gene sequencing, polymerase chain reaction, isothermal amplification reaction, gene chip, probe hybridization, gel electrophoresis, Northern blotting, nucleic acid mass spectrometry, single molecule detection or liquid chromatography.

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

[0025] Furthermore, the protein expression level is measured by amino acid sequencing, enzyme-linked immunosorbent assay, chemiluminescence, immunochromatography, radioimmunoassay, immunohistochemistry, immunoblotting, flow cytometry, gel electrophoresis, protein profiling, immuno-PCR, single molecule detection or liquid chromatography.

[0026] In some aspects, the reagents also include reagents for detecting the expression levels of other markers in the sample.

[0027] Furthermore, the other markers include one or more of HES4, EIF2AK2, TTC21A, OTOF and ANKRD22.

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

[0029] In some aspects, the product further comprises a standard, a calibrator, a control, or a buffer.

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

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

[0032] In some aspects, the diagnosis includes but is not limited to screening for Sjögren's syndrome, 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 Sjögren's syndrome or distinguishing Sjögren's syndrome from systemic lupus erythematosus, including a detection agent or component for obtaining the expression level of ZCCHC2 in a sample. Specifically, a product for diagnosing Sjögren's syndrome; specifically, a product for distinguishing Sjögren's syndrome from systemic lupus erythematosus.

[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 ZCCHC2 expression level can be used independently as an indicator for the diagnosis of Sjögren's syndrome.

[0036] In some aspects, the ZCCHC2 expression level serves as an independent indicator for distinguishing Sjögren's syndrome from systemic lupus erythematosus.

[0037] In some aspects, the expression level comprises a nucleic acid expression level or a protein expression level.

[0038] In some aspects, when the expression level is a nucleic acid expression level, the reagent is suitable for detecting the nucleic acid expression level by sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology.

[0039] In some aspects, when the expression level is a protein expression level, the reagent for detecting the protein expression level is suitable for detecting the protein expression level by sequencing technology, immunological technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology.

[0040] In some aspects, the nucleic acid expression level or protein expression level includes but is not limited to the abundance or concentration of the nucleic acid or protein; in some aspects, it may also include the mutation abundance or concentration of the nucleic acid or protein.

[0041] Furthermore, the nucleic acid expression level is obtained by sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology.

[0042] Furthermore, the nucleic acid expression level is obtained or detected by gene sequencing, polymerase chain reaction, isothermal amplification reaction, gene chip, probe hybridization, gel electrophoresis, Northern blotting, nucleic acid mass spectrometry, single molecule detection or liquid chromatography.

[0043] Furthermore, the protein expression level is obtained by sequencing technology, immunoassay technology, electrophoresis technology, mass spectrometry technology or chromatography technology.

[0044] Furthermore, the method for obtaining the protein expression level 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, immuno-PCR, single molecule detection or liquid chromatography.

[0045] In some aspects, the reagents also include reagents for detecting the expression levels of other markers in the sample.

[0046] Furthermore, the other markers include one or more of HES4, EIF2AK2, TTC21A, OTOF and ANKRD22.

[0047] In some aspects, the product further includes a sample processing reagent, and the sample processing reagent includes a sample lysis reagent, a sample purification reagent, or a sample extraction reagent.

[0048] In some aspects, the product further comprises a standard, a calibrator, a control, or a buffer.

[0049] In some aspects, the sample comprises one or more of tissue, cells, body fluids, serum, plasma, whole blood (peripheral blood), urine, semen, saliva, pleural effusion, ascites, cerebrospinal fluid, stool, and synovial fluid.

[0050] In some aspects, the sample is peripheral blood mononuclear cells.

[0051] In some aspects, the diagnosis includes but is not limited to screening for Sjögren's syndrome, assisting in diagnosis, confirming the disease, predicting / judging the severity of the disease, assessing disease activity, and monitoring treatment.

[0052] The present application also provides a method for diagnosing Sjögren's syndrome in vivo or in vitro, comprising the step of obtaining the expression level of ZCCHC2 in a sample from a subject.

[0053] Further, the method comprises the following steps: (S1) detecting or determining the expression level of ZCCHC2 in the subject sample; (S2a) comparing the ZCCHC2 expression level with that of a control sample; wherein a significant difference in the level of ZCCHC2 between the subject sample and the control sample is an indication that the subject suffers from Sjögren's syndrome; or; (S2b) comparing with a set threshold absolute amount; wherein the ZCCHC2 expression level of the subject sample higher than the threshold absolute amount is an indication that the subject suffers from Sjögren's syndrome; or; (S2c) using the ZCCHC2 expression level in the subject sample as an input value, operating in an algorithm for determining Sjögren's syndrome, and determining whether the subject suffers from Sjögren's syndrome.

[0054] In some aspects, the ZCCHC2 can be used independently as an indicator for the diagnosis of Sjögren's syndrome.

[0055] The present application also provides a method for distinguishing Sjögren's syndrome and systemic lupus erythematosus in vivo or in vitro, characterized in that it includes a step of obtaining the expression level of ZCCHC2 in a sample from a subject.

[0056] Further, the method comprises the following steps: (S1) detecting or determining the expression level of ZCCHC2 in the sample of the subject; (S2a) comparing with the expression level of ZCCHC2 in the control sample; wherein, a significant difference in the expression level of ZCCHC2 between the sample and the control sample is an indication that the subject suffers from Sjögren's syndrome; or; (S2b) comparing with a set threshold absolute amount; wherein, the threshold absolute amount can distinguish between Sjögren's syndrome and systemic lupus erythematosus; in some aspects, the expression level of ZCCHC2 in the sample of the Sjögren's syndrome patient is higher than the expression level of ZCCHC2 in the sample of the systemic lupus erythematosus patient; or; (S2c) using the expression level of ZCCHC2 in the sample of the subject as an input value and operating in an algorithm for distinguishing Sjögren's syndrome from systemic lupus erythematosus; and determining whether the subject suffers from Sjögren's syndrome rather than systemic lupus erythematosus.

[0057] In some aspects, the ZCCHC2 expression level serves as an independent indicator for distinguishing Sjögren's syndrome from systemic lupus erythematosus.

[0058] The present application also provides a method for detecting a marker in a subject suffering from or suspected of suffering from Sjögren's syndrome in vivo or in vitro, the method comprising determining or detecting the expression level of ZCCHC2 in a sample from the subject.

[0059] In some aspects, the method further comprises determining or detecting the expression level of one or more of HES4, EIF2AK2, TTC21A, OTOF, and ANKRD22 in a sample from the subject.

[0060] The present application may also provide a method for evaluating or screening diagnostic markers for Sjögren's syndrome, comprising the step of performing correlation or consistency analysis on potential biomarkers and ZCCHC2 expression levels, and verifying the diagnostic value of potential markers through the conclusion of correlation or consistency.

[0061] The present application also provides a method for in vivo or in vitro screening of a therapeutic agent for Sjögren's syndrome, the method comprising the step of evaluating the expression level of ZCCHC2 in a sample treated with the therapeutic agent.

[0062] The present application also provides a marker for diagnosing Sjögren's syndrome, the marker comprising ZCCHC2 protein or a nucleic acid encoding ZCCHC2 protein; preferably, the marker further comprises one or more of HES4, EIF2AK2, TTC21A, OTOF and ANKRD22 proteins, or one or more of nucleic acids encoding HES4, EIF2AK2, TTC21A, OTOF and ANKRD22 proteins.

[0063] In some aspects of the above methods, the expression level includes a nucleic acid expression level or a protein expression level.

[0064] In some aspects of the above methods, when the expression level is a nucleic acid expression level, the reagent is suitable for detecting the nucleic acid expression level by sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology.

[0065] In some aspects of the above method, when the expression level is a protein expression level, the reagent for detecting the protein expression level is suitable for detecting the protein expression level by sequencing technology, immunological technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology.

[0066] In some aspects of the above methods, the nucleic acid expression level or protein expression level includes but is not limited to the abundance or concentration of the nucleic acid or protein; in some aspects, it may also include such as the mutation abundance or concentration of the nucleic acid or protein.

[0067] In some aspects of the above methods, the nucleic acid expression level is obtained or detected by sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology.

[0068] In some aspects of the above methods, the nucleic acid expression level is obtained or detected by gene sequencing, polymerase chain reaction, isothermal amplification reaction, gene chip, probe hybridization, gel electrophoresis, Northern blotting, nucleic acid mass spectrometry, single molecule detection or liquid chromatography.

[0069] In some aspects of the above methods, the protein expression level is obtained or detected by sequencing technology, immunoassay technology, electrophoresis technology, mass spectrometry technology or chromatography technology.

[0070] In some aspects of the above methods, the protein expression level is obtained or detected by amino acid sequencing, enzyme-linked immunosorbent assay, chemiluminescence, immunochromatography, radioimmunoassay, immunohistochemistry, immunoblotting, flow cytometry, gel electrophoresis, protein profiling, immuno-PCR, single molecule detection or liquid chromatography.

[0071] In some aspects of the above method, the method further comprises the step of obtaining the expression levels of other markers in the subject's sample.

[0072] In some aspects of the above methods, the other markers include one or more of HES4, EIF2AK2, TTC21A, OTOF, and ANKRD22.

[0073] In some aspects of the above methods, the sample includes one or more of tissue, cells, body fluids, serum, plasma, whole blood (peripheral blood), urine, semen, saliva, pleural effusion, ascites, cerebrospinal fluid, feces and synovial fluid.

[0074] In some aspects of the above methods, the sample is peripheral blood mononuclear cells.

[0075] In some aspects of the above methods, the diagnosis includes but is not limited to screening for Sjögren's syndrome, auxiliary diagnosis, diagnosis, prediction / determination of disease severity, assessment of disease activity, and monitoring of treatment.

[0076] In some aspects of the above methods, the subject is preferably a human.

[0077] In some aspects of the above methods, the methods can be used for disease diagnosis or non-disease diagnosis purposes.

[0078] The present application also provides a method for treating Sjögren's syndrome, the method comprising the following steps:

[0079] (1) Obtaining the ZCCHC2 expression level in the subject sample and comparing it with the ZCCHC2 expression level in the control sample;

[0080] (2) If the ZCCHC2 expression level in the subject's sample is compared with the set threshold absolute amount, the subject is given glucocorticoids, antimalarial drugs, immunosuppressants, immunoglobulins, biologics, or nonsteroidal anti-inflammatory drugs.

[0081] In some aspects, the antimalarial drugs include but are not limited to hydroxychloroquine.

[0082] In some aspects, the immunosuppressant includes, but is not limited to, one or more of cyclophosphamide, methotrexate, azathioprine, cyclosporine, mycophenolate mofetil, cyclosporine, levelast, pilocarpine, and cevimeline.

[0083] In some aspects, the biologic includes but is not limited to an anti-CD20 antibody.

[0084] Furthermore, the CD20 antibody includes but is not limited to one or more of rituximab, ibritumomab, ofatumumab, ocrelizumab, veltuzumab, and tositumomab.

[0085] Beneficial technical effects of this application:

[0086] This application screens genes or their proteins that are differentially expressed in peripheral blood monocytes of Sjögren's syndrome patients and normal people, and for the first time proposes that the ZCCHC2 gene or protein can be used as a biomarker for the diagnosis of Sjögren's syndrome. It can also be used as a biomarker for distinguishing Sjögren's syndrome from systemic lupus erythematosus, improve the misdiagnosis of Sjögren's syndrome, make up for the shortcomings of the existing diagnosis of Sjögren's syndrome, and has significant clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] 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.

[0088] Figure 1 , The results of discovery set analysis showed that the gene ZCCHC2 was upregulated in peripheral blood mononuclear cells (PBMCs) of patients with Sjögren's syndrome;

[0089] Figure 2 , The ROC curve analysis results of the discovery set showed that the gene had a good ability to distinguish patients from normal people;

[0090] Figure 3 , the results of validation set analysis showed that the expression of gene ZCCHC2 was increased in peripheral blood mononuclear cells (PBMC) of patients with Sjögren's syndrome;

[0091] Figure 4 , The ROC curve analysis results of the validation set showed that the gene had good discrimination between patients and normal people;

[0092] Figure 5 ,The results of logistic regression analysis revealed that the AUC value of ZCCHC2 combined with HES4, TTC21A, EIF2AK2, OTOF and ANKRD22 in the discovery set data can reach 0.878;

[0093] Figure 6 , in the discovery set data, when ZCCHC2 was used in combination with HES4, EIF2AK2, TTC21A, OTOF, and ANKRD22, the AUC values ​​could be increased to 0.830, 0.852, 0.797, 0.814, and 0.806, respectively;

[0094] Figure 7 , the expression of ZCCHC2 in PBMCs of patients with systemic lupus erythematosus (SLE) was not significantly increased (fold difference = 1.3), indicating that ZCCHC2 has a good ability to distinguish SLE from SS;

[0095] Figure 8 , Clinical samples verified that the expression level of ZCCHC2 in PBMCs of patients with Sjögren's syndrome was significantly increased;

[0096] Fig. 9 , the ROC curve analysis results were verified by clinical samples, and the AUC data showed that ZCCHC2 had a good performance in diagnosing Sjögren's syndrome. DETAILED DESCRIPTION OF THE INVENTION

[0098] The content for which protection is requested in this application can be realized by those skilled in the art with reference to the content of this article. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application. The methods and applications of this application have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the preparation methods and applications herein 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 meanings as those of ordinary skill in the art to which this application belongs.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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%.

[0103] 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.

[0104] 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.

[0105] Diagnostic and differential use

[0106] This application screened out the protein ZCCHC2 that is differentially expressed in peripheral blood mononuclear cells of patients with Sjögren's syndrome and normal people through the discovery set, and drew the receiver operating characteristic (ROC) curve of ZCCHC2 in the discovery data set. It was found that the area under the ROC curve (Area Under Curve, AUC) value of ZCCHC2 in the discovery set was 0.714, indicating that it has good performance in distinguishing patients with Sjögren's syndrome from normal people. The validation set analysis also found that ZCCHC2 was significantly expressed in peripheral blood mononuclear cells of patients with Sjögren's syndrome; at the same time, the ROC curve analysis also found that the AUC value of ZCCHC2 can reach 0.765. Surprisingly, the expression difference multiple of ZCCHC2 in patients with Sjögren's syndrome is significantly higher than the differential expression multiple in patients with systemic lupus erythematosus, so ZCCHC2 can achieve the identification of Sjögren's syndrome and systemic lupus erythematosus.

[0107] Therefore, in various aspects of the present disclosure, provided is the use of a detection agent or component for obtaining the expression level of ZCCHC2 in a sample in the preparation of a diagnostic product, wherein the diagnostic product includes a product for diagnosing Sjögren's syndrome or a product for distinguishing Sjögren's syndrome from systemic lupus erythematosus; or; the use of ZCCHC2 expression level as a marker in diagnosing Sjögren's syndrome or distinguishing Sjögren's syndrome from systemic lupus erythematosus.

[0108] As used herein, the terms "Sjögren's syndrome" and "SS" are interchangeable. Overview of Sjögren's syndrome Sjögren's syndrome is a chronic autoimmune disease that mainly invades exocrine glands such as lacrimal glands and salivary glands, also known as autoimmune exocrine gland disease. It is mainly manifested as keratoconjunctivitis sicca, conjunctivitis, xerostomia or other rheumatic diseases such as rheumatoid arthritis. It can involve other systems such as respiratory system, digestive system, urinary system, blood system, nervous system, muscles, joints, etc., causing multi-system and multi-organ damage. This disease can exist alone or in other autoimmune diseases. The one that exists alone is primary Sjögren's syndrome, while the one that is secondary to other autoimmune diseases such as rheumatoid arthritis, systemic sclerosis, systemic lupus erythematosus is secondary Sjögren's syndrome. The disease has a high incidence rate and is more common in women over 40 years old. Its pathological mechanism is mainly due to the excessive response of autoimmunity, which causes a large number of lymphocytes and plasma cells to infiltrate the exocrine glands, destroy the glandular cells, and lose their function, resulting in a series of clinical symptoms and manifestations. Sjögren's syndrome is a global disease. More than 90% of cases occur in women. Most cases occur in middle-aged and elderly people aged 40 to 60, and there are fewer cases in children. Due to the lack of a unified diagnostic standard, the prevalence of the disease is very inaccurate. It is generally estimated to be 0.1% to 0.7%. In the United States, the incidence of Sjögren's syndrome is second only to rheumatoid arthritis.

[0109] 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.

[0110] As used herein, the term "ZCCHC2" (Zinc Finger CCHC-Type Containing 2) is a protein coding gene. Diseases associated with ZCCHC2 include familial expansive osteolysis. Gene ontology (GO) annotations associated with this gene include nucleic acid binding and phosphatidylinositol binding.

[0111] As used herein, the term "diagnosis" refers to the process of identifying a medical condition or disease (such as SS) through its signs, symptoms, and especially the results of various diagnostic processes (including detecting the expression level of ZCCHC2 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 a disease exists), predicting or determining the severity of a disease, evaluating disease activity, monitoring treatment, such as monitoring disease deterioration or recurrence during treatment, evaluating the efficacy of disease treatment, and the selection of a given treatment regimen. Without limitation, in some specific embodiments, the present application has fully demonstrated that the expression level of ZCCHC2 can be used to determine whether a subject has SS, and can then be used for screening, auxiliary diagnosis, and diagnosis; in other specific embodiments, it is demonstrated that the expression level of ZCCHC2 can be used to predict or determine the severity of SS, evaluate disease activity, and can be used to monitor patients after treatment, and determine whether the condition has improved by severity or activity. As used herein, the term "differentiation" refers to differentiating different diseases associated with clinical manifestations (e.g., differentiating Sjögren's syndrome from systemic lupus erythematosus) through various diagnostic processes (e.g., obtaining in vitro samples of individuals for marker level detection, such as ZCCHC expression level detection), in order to improve misdiagnosis and improve diagnostic accuracy. In some specific embodiments, the differential expression fold of ZCCHC2 in Sjögren's syndrome patients is significantly higher than the differential expression fold in systemic lupus erythematosus patients, confirming that ZCCHC2 expression levels can be used to differentiate Sjögren's syndrome from systemic lupus erythematosus.

[0112] As used herein, "level" is understood in the broadest sense, including qualitative or quantitative levels, including but not limited to content, abundance, concentration, degree, ratio, multiple, etc.

[0113] 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.

[0114] 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.

[0115] As used herein, the terms "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 SS 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. In a specific embodiment, the sample includes peripheral blood mononuclear cells. 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 in culture, cell supernatants, tissue samples, organs, etc. 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.

[0116] 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.

[0117] 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 the method and can provide a measure of the diagnostic utility of the method. Typically, ROC-AUC is in the range of 0.5 to 1.0, where values ​​closer to 0.5 indicate that the method has limited diagnostic utility (e.g., lower sensitivity and / or specificity), and values ​​closer to 1.0 indicate 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.

[0118] The detection agent or component for obtaining the expression level of ZCCHC2 in the sample can be understood to include a detection agent for directly obtaining the expression level of ZCCHC2 in the sample, or a component for indirectly obtaining the expression level of ZCCHC2 in the sample (such as a computer program directly obtaining an indicator of the expression level of ZCCHC2 in a sample that has been detected).

[0119] According to some embodiments of the present application, marker level indicators can be obtained in any manner such as nucleic acid expression level, protein expression level, etc. The detection method is not limited, and any method that can be used to directly or indirectly evaluate the expression level of ZCCHC2 or other marker nucleic acids or proteins is suitable for the present application.

[0120] As used herein, "nucleic acid expression level" should be understood in a broad sense, including both DNA level and RNA level (such as free RNA); the level includes but is not limited to methylation, copy number, variation, and expression level.

[0121] 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.

[0122] 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.

[0123] For another example, in some specific embodiments, the polymerase chain reaction method includes primers for ZCCHC2 amplification, wherein the primer refers to a primer that can specifically amplify the ZCCHC2 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 ZCCHC2, wherein the probe refers to a probe that can specifically identify the ZCCHC2 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 specific sequence of ZCCHC2 as a target nucleotide) through complementary base pairing. In some specific embodiments, the 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 ZCCHC2 amplification.

[0124] 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, immune PCR, single molecule detection or liquid chromatography.

[0125] For example, in some embodiments, the method for quantitatively detecting ZCCHC2 at the protein expression level includes an antibody. In a preferred embodiment, the antibody is a specific antibody. Among them, the specific antibody refers to an antibody that can specifically recognize the protein encoded by ZCCHC2, 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.

[0126] According to the experimental data of the present application, the ZCCHC2 can be used as an independent indication for the diagnosis of Sjögren's syndrome. An effective diagnosis of Sjögren's syndrome can be achieved based on ZCCHC2 alone. Unexpectedly, ZCCHC2 can also be used as an independent indication for differentiating Sjögren's syndrome from systemic lupus erythematosus, thereby improving the misdiagnosis of Sjögren's syndrome.

[0127] It is understandable that in order to further enhance the diagnostic effect of Sjögren's syndrome, 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., 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 cooperate with each other to achieve a better diagnostic effect on patients with Sjögren's syndrome, thereby achieving a more effective diagnostic evaluation. Therefore, in addition to being an independent indication for the diagnosis of Sjögren's syndrome, the ZCCHC2 can also be detected in combination with other existing known diagnostic markers to improve the diagnostic efficiency.

[0128] For example, in some specific embodiments, the diagnosis also includes diagnostic reagents for detecting other genes or proteins, such as HES4, EIF2AK2, TTC21A, OTOF and ANKRD22 mentioned in this application. The combined detection can be a combination of any one or more of them. In a preferred embodiment of the present application, when ZCCHC2 and HES4, EIF2AK2, TTC21A, OTOF and ANKRD22 are used simultaneously for the diagnosis and evaluation of Sjögren's syndrome, it has a higher AUC value of 0.878, which significantly improves the effect of distinguishing patients with Sjögren's syndrome from normal people.

[0129] In some embodiments of the present application, when ZCCHC2 is combined with HES4, EIF2AK2, TTC21A, OTOF and ANKRD22 in pairs, the AUC value is also improved, thereby enhancing the effect of distinguishing patients with Sjögren's syndrome from normal people.

[0130] 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 sample is selected from any one of tissues, cells, serum, plasma, whole blood, urine, semen, and saliva.

[0131] Diagnosis and Differential Approach

[0132] The applicant found that the level of ZCCHC2 in peripheral blood monocytes of patients with Sjögren's syndrome is higher than that in peripheral blood monocytes of healthy people. Therefore, a more accurate prediction of the diagnosis of Sjögren's syndrome can be made based on the level of ZCCHC2 in peripheral blood monocytes (such as expression level).

[0133] The core of the diagnostic method includes the steps of detecting or determining the expression level of ZCCHC2 in a sample from a subject;

[0134] In some specific embodiments, the method comprises the following steps:

[0135] (S1) detecting or determining the level of ZCCHC2 in the test sample;

[0136] (S2a) comparing the expression level of ZCCHC2 with that of a control sample, wherein a significant difference in the expression level of ZCCHC2 between the test sample and the control sample is an indication that the subject suffers from Sjögren's syndrome; or;

[0137] (S2b) 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 Sjögren's syndrome; or; (S2c) using the ZCCHC2 expression level in the subject's sample as an input value, calculating it in an algorithm for determining Sjögren's syndrome, and determining whether the subject suffers from Sjögren's syndrome.

[0138] The applicant also found that the differential expression multiple of ZCCHC2 in patients with Sjögren's syndrome was significantly higher than that in patients with systemic lupus erythematosus, and ZCCHC2 can be used to differentiate between Sjögren's syndrome and systemic lupus erythematosus and improve misdiagnosis.

[0139] The present application also provides a method for distinguishing Sjögren's syndrome and systemic lupus erythematosus in vivo or in vitro, characterized in that it includes a step of obtaining the expression level of ZCCHC2 in a sample from a subject.

[0140] In some specific embodiments, the method comprises the following steps: (S1) detecting or determining the expression level of ZCCHC2 in the sample of the subject; (S2a) comparing with the expression level of ZCCHC2 in the control sample; wherein a significant difference in the expression level of ZCCHC2 between the sample and the control sample is an indication that the subject suffers from Sjögren's syndrome; or; (S2b) comparing with a set threshold absolute amount; wherein the threshold absolute amount can distinguish Sjögren's syndrome from systemic lupus erythematosus, and in some aspects the expression level of ZCCHC2 in the sample of the Sjögren's syndrome patient is higher than the expression level of ZCCHC2 in the sample of the systemic lupus erythematosus patient; or; (S2c) using the expression level of ZCCHC2 in the sample of the subject as an input value and operating in an algorithm for distinguishing Sjögren's syndrome from systemic lupus erythematosus.

[0141] As used herein, "algorithms" include any equation, model, mathematical formula, algorithmic, analytical or programmed process or statistical technique or classification analysis that takes one or more input data or parameters (whether continuous or categorical) and calculates an output value, index, index value or score. Examples of algorithms include, but are not limited to, ratios, sums, regression operators such as indices or coefficients, biomarker value conversion and standardization (including but not limited to standardized models based on clinical parameters such as age, gender, race, etc.), rules and criteria, statistical classification models, and neural networks trained on populations. In some embodiments, such as in the case of inputting a subject's biomarker level, the algorithm can be a linear or nonlinear equation and statistical classification analysis of the relationship between the subject's marker level and disease diagnosis, identification, etc., and the algorithm can be implemented based on the input value, obtain an output value, and determine whether the subject is in a disease state based on the output value.

[0142] In some embodiments, the "level of ZCCHC2" herein includes but is not limited to the abundance or concentration of ZCCHC2 nucleic acid or protein, such as in some specific embodiments, the expression level or concentration of the corresponding protein.

[0143] 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.

[0144] In some specific embodiments, a set value of the expression level of ZCCHC2 can be given, and the set value can be determined according to the expression level of ZCCHC2 of normal samples of normal people and / or non-Sjögren's syndrome patients. For example, the average value or median value of the expression level of ZCCHC2 of normal samples of an appropriate number of samples is selected, or a reasonable multiple is set based on the average value, such as more than 2 times, more than 1.9 times, more than 1.8 times, more than 1.7 times, more than 1.6 times, more than 1.5 times, more than 1.4 times, more than 1.3 times, more than 1.2 times, more than 1.1 times, more than 1 times, when the expression level of ZCCHC2 of the subject is higher than the set value, it is judged to be Sjögren's syndrome. It can be understood 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. The "threshold" described herein is based on the conventional understanding in the art. In the process of disease diagnosis and identification, a set cut-off is given to achieve a series of diagnosis and identification of the disease. For example, some thresholds are set with reference to AUC. In some specific embodiments, the absolute value range of the threshold for diagnosing Sjögren's syndrome is 4.05-5.65, that is, the expression level of ZCCHC2 in the sample is higher than the absolute value of the threshold (higher than any value in the range of 4.05-5.65), which is an indication that the subject suffers from Sjögren's syndrome.

[0145] In some specific embodiments, a set value of the expression level of ZCCHC2 can be given, and the set value can be determined according to the expression level of ZCCHC2 in samples of patients with systemic lupus erythematosus. For example, the average value or median value of the expression level of ZCCHC2 of systemic lupus erythematosus of a suitable number of samples is selected, or a reasonable multiple is set based on the average value, such as more than 2 times, more than 1.9 times, more than 1.8 times, more than 1.7 times, more than 1.6 times, more than 1.5 times, more than 1.4 times, more than 1.3 times, more than 1.2 times, more than 1.1 times, more than 1 times. When the expression level of ZCCHC2 of the subject is higher than the set value, it is judged to be Sjögren's syndrome rather than systemic lupus erythematosus. It can be understood that the set value determined based on the average value or median value, or the multiple of the average value or median 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. For example, in some specific embodiments, when the threshold is set based on a difference fold greater than 1.3 or even higher (such as 1.4, 1.5, 1.6, 1.7, or 1.8), ZCCHC2 can achieve the differentiation between Sjögren's syndrome and systemic lupus erythematosus.

[0146] The "threshold" described herein is based on the common understanding in the art. In the process of disease diagnosis and identification, a set distinction value (cut-off) is given to achieve a series of diagnosis and identification of diseases. For example, some thresholds are set with reference to AUC.

[0147] Wherein, the level of ZCCHC2 refers to a value derived by direct measurement or further indirect derivation based on direct measurement for this biomarker of the subject, 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.

[0148] product

[0149] According to the core diagnostic use of this application, the process for detecting the expression level of ZCCHC2 can be configured into a corresponding product form for the diagnosis of Sjögren's syndrome or differentiation of systemic lupus erythematosus. The product includes a reagent or component for detecting the ZCCHC2 biomarker. It is understood that such product forms are diverse, including but not limited to a kit form, a system device, a computer-readable medium, or a computer system form.

[0150] Kit Format

[0151] In some embodiments of the present application, a kit for detecting or analyzing the expression level of ZCCHC2 to diagnose SS disease or differentiate SS from SLE is also disclosed herein. Such a kit may include reagents for detecting the level of one or more markers and instructions for predicting SS disease or distinguishing SS from SLE based on the detected levels.

[0152] 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 ZCCHC2 expression levels. The set of reagents may also further detect one or more other marker levels. In some aspects, the reagents are detected at the level of nucleic acid or protein.

[0153] When the detection agent is used to detect nucleic acid expression levels, 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.

[0154] 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 ZCCHC2 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 other embodiments, the reagent is a probe reagent, wherein the probe refers to a probe capable of specifically identifying the ZCCHC2 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 ZCCHC2 as a target nucleotide) through complementary base pairing.

[0155] When the detection agent is used to detect protein expression levels, it can be understood that there are many protein detection methods in the art, including but not limited to sequencing technology, immunoassay 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, radioimmunoassay, immunohistochemistry, immunoblotting, flow cytometry, gel electrophoresis, protein spectrometry or liquid chromatography.

[0156] Thus, for example, in some embodiments, the reagent is an antibody. In a specific embodiment, the reagent is a specific antibody, wherein the specific antibody refers to an antibody that can specifically recognize the protein encoded by ZCCHC2, 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] System Devices

[0163] In some embodiments of the present application, a system for detecting or analyzing ZCCHC2 expression levels to predict SS disease or distinguish SS from SLE 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 ZCCHC2 expression level; and a computer system coupled to the device to obtain the measured level and determine a score for predicting the possibility of SS disease in SS patients.

[0164] The device is configured to detect the expression level of ZCCHC2 in a mixture of a reagent and a test sample. For example, the device can determine the expression level of ZCCHC2 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 wells, 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 the device include an ELISA reader (e.g., a luminescent ELISA reader, an absorbance ELISA reader, a fluorescence ELISA reader), a spectrometer and a spectrophotometer.

[0165] 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 predictive model and determines the likelihood of a SS disease activity event in the subject.

[0166] Computer system or computer readable medium

[0167] 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 expression level of ZCCHC2 by a computer to predict SS disease.

[0168] For example, the construction and execution of the prediction model for generating a score (e.g., LFPI score) can be implemented 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 a machine programmed with instructions utilizing the data is used, the data storage material can display any data set and execution and results of the prediction model of the present application. Such data can be used for a variety of purposes, such as SS patient monitoring, diagnosis, treatment considerations, etc. The embodiment 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. 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 conventional design.

[0169] 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.

[0170] 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.

[0171] Other aspects

[0172] It can be understood that on the basis of believing that ZCCHC2 can be used as a diagnostic marker for Sjögren's syndrome, those skilled in the art can evaluate or screen other potential diagnostic markers for Sjögren's syndrome based on this, and usually evaluate the correlation between potential biomarkers and ZCCHC2, and on the basis of reaching a positive correlation conclusion, it helps to illustrate the value of the potential diagnostic marker.

[0173] The present application may also relate to a method for detecting a marker in a subject suffering from or suspected of suffering from Sjögren's syndrome in vivo or in vitro, the method comprising determining or detecting the level of ZCCHC2 in a biological sample from the subject, and the method may include uses for non-disease diagnosis.

[0174] In addition, the present application may also relate to a method for in vivo or in vitro screening of a therapeutic agent for Sjögren's syndrome, the method comprising the step of evaluating the ZCCHC2 expression level of a sample treated with a therapeutic agent, and evaluating the therapeutic effect of the therapeutic agent based on the ZCCHC2 expression level. For example, when the ZCCHC2 expression level decreases, the therapeutic effect of the therapeutic agent can be determined.

[0175] The implementation scheme of the present application is described in detail below with reference to examples.

[0176] Dataset

[0177] This application downloads three expression matrix files of peripheral blood mononuclear cell samples of Sjögren's syndrome patients from the Gene Expression Omnibus (GEO) database, namely GSE51092, GSE66795, and GSE140161. Among them, the GSE51092 data set contains 191 SS and 33 control samples (normal people), the GSE66795 data set contains 132 SS and 30 control samples (normal people), and the GSE140161 data set contains 352 SS samples. After merging and normalizing the GSE24706, GSE61635, and GSE65391 data, all samples were allocated as discovery sets and validation sets in a ratio of 7:3.

[0178] Example 1. Analysis of the expression of ZCCHC2 in peripheral blood mononuclear cells (PBMC) of patients with Sjögren's syndrome

[0179] In this example, 517 SS samples and 39 control samples (normal people) 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 ZCCHC2 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. ZCCHC2 was also screened out. The results are as follows: Figure 1 As shown, the gene ZCCHC2 is upregulated in peripheral blood mononuclear cells (PBMC) of patients with Sjögren's syndrome, confirming that ZCCHC2 is a diagnostic marker for Sjögren's syndrome.

[0180] Example 2: Evaluation and analysis based on ROC curve

[0181] In order to test the diagnostic efficacy of the candidate markers, the present embodiment plotted the receiver operating characteristic (ROC) curve of ZCCHC2 in the metadata cohort. The results showed that the area under the ROC curve (AUC) value of ZCCHC2 in the discovery set was 0.714 (see Figure 2 ), indicating that ZCCHC2 has good performance in distinguishing patients with Sjögren's syndrome from normal people.

[0182] Example 3: Verification of the verification set

[0183] In this example, a statistical analysis was performed on a data set of 157 SS samples and 23 control samples (normal subjects), and it was found that the expression of ZCCHC2 in peripheral blood mononuclear cells of patients with Sjögren's syndrome was also significantly increased (see Figure 3 ); At the same time, ROC curve analysis in this cohort found that the AUC of ZCCHC2 could reach 0.765 (see Figure 4 ), which fully proves that ZCCHC2 has good performance in diagnosing Sjögren's syndrome.

[0184] Example 4: Evaluation of combined use with existing Sjögren's syndrome diagnostic markers

[0185] This example evaluates whether ZCCHC2 has enhanced detection efficacy when used in combination with known Sjögren's syndrome markers. ZCCHC2 was combined with five Sjögren's syndrome diagnostic markers HES4, EIF2AK2, TTC21A, OTOF, and ANKRD22, and the effect of the combination in distinguishing Sjögren's syndrome from normal people was tested (AUC value evaluation). Similarly, subsequent analysis was performed in the metadata queue of Example 1.

[0186] The experiment found that when ZCCHC2 was combined with HES4, EIF2AK2, TTC21A, OTOF, and ANKRD22, the AUC value was 0.878 (see Figure 5 ); When ZCCHC2 was combined with HES4, EIF2AK2, TTC21A, OTOF, and ANKRD22, the AUC values ​​were increased to 0.830, 0.852, 0.797, 0.814, and 0.806, respectively (see Figure 6 ); It can be seen that the combined use improves the differentiation effect between Sjögren's syndrome patients and normal people, further confirming the diagnostic value of ZCCHC2.

[0187] Example 5: Discrimination of different autoimmune diseases

[0188] Some patients with Sjögren's syndrome have clinical symptoms of systemic lupus erythematosus, such as skin erythema, so there is a possibility of misdiagnosis. In the GEO combined data set (GSE24706, GSE61635, GSE65391), experiments found that ZCCHC2 expression in PBMC of Sjögren's syndrome patients was higher than that in normal people (discovery set difference fold = 1.8 times) (see Figure 1 , 3 ), this application downloaded three expression matrix files of peripheral blood mononuclear cell samples of patients with systemic lupus erythematosus from the Gene Expression Omnibus (GEO) database, namely GSE65391, GSE121239 and GSE61635 to construct a discovery set, and randomly selected 945 SLE and 85 control samples to analyze the expression of ZCCHC2, and found that it was not significantly increased in PBMC of patients with systemic lupus erythematosus (difference fold = 1.3 times) (see Figure 7 ). Therefore, the higher the expression of ZCCHC2, the more likely the patient is suffering from Sjögren's syndrome rather than systemic lupus erythematosus. Therefore, ZCCHC2 can be used to differentiate between Sjögren's syndrome and systemic lupus erythematosus.

[0189] Example 6: Clinical sample testing

[0190] In this example, 24 samples of human whole blood were collected, including 12 samples from Sjögren's syndrome patients and 12 samples from healthy subjects. After all the whole blood specimens were collected, peripheral blood mononuclear cells were separated, 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), and the detection primers were designed and synthesized by a third-party company. The relative expression of the gene was calculated using the 2-ΔCT method. A p value <0.05 was considered statistically significant. The test results showed that there was a statistical difference in the expression level of ZCCHC2 between patients with Sjögren's syndrome and healthy subjects (see Figure 8 ); ROC curve analysis showed that the AUC of ZCCHC2 in clinical diagnosis of Sjögren's syndrome was 0.760 (see Fig. 9), with a specificity of 66.7% and a sensitivity of 90.9%. Therefore, ZCCHC2 has a good clinical application value in the diagnosis of Sjögren's syndrome.

[0191] 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 reagent for detecting the expression level of ZCCHC2 in a sample in the preparation of a diagnostic product, wherein the diagnostic product is a product for diagnosing Sjögren's syndrome or a product for distinguishing Sjögren's syndrome from systemic lupus erythematosus.

2. The use according to claim 1, wherein: The expression level includes nucleic acid expression level or protein expression level; preferably, when the expression level is nucleic acid expression level, the reagent is suitable for detecting the nucleic acid expression level by sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology; when the expression level is protein expression level, the reagent for protein expression level is suitable for detecting the protein expression level by sequencing technology, immune technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology.

3. The use according to claim 1 or 2, wherein: The reagents also include reagents for detecting the expression levels of other markers in a sample; the other markers include one or more of HES4, EIF2AK2, TTC21A, OTOF and ANKRD22.

4. The use according to any one of claims 1 to 3, wherein: The sample includes one or more of tissue, cells, body fluid, serum, plasma, whole blood, urine, semen, saliva, pleural effusion, ascites, cerebrospinal fluid, feces and synovial fluid.

5. A method for diagnosing Sjögren's syndrome, wherein: The method comprises the steps of obtaining the expression level of ZCCHC2 in the sample; Optionally, the method comprises the following steps: (S1) detecting or determining the expression level of ZCCHC2 in the sample; (S2a) comparing the expression level of ZCCHC2 in the sample with that in the control sample; wherein a significant difference in the expression level of ZCCHC2 between the sample and the control sample is an indication that the subject suffers from Sjögren's syndrome; or; (S2b) comparing with a set threshold absolute amount; wherein the expression level of ZCCHC2 in the sample being higher than the threshold absolute amount is an indication that the subject suffers from Sjögren's syndrome; or; (S2c) using the expression level of ZCCHC2 in the sample as an input value, calculating it in an algorithm for determining Sjögren's syndrome, and determining whether the subject suffers from Sjögren's syndrome; Preferably, the method further comprises the step of acquiring the expression levels of other markers in the sample, wherein the other markers include one or more of HES4, EIF2AK2, TTC21A, OTOF and ANKRD22.

6. A method for distinguishing Sjögren's syndrome from systemic lupus erythematosus, wherein: The method comprises the steps of obtaining the expression level of ZCCHC2 in the sample; Optionally, the method comprises the following steps: (S1) detecting or determining the expression level of ZCCHC2 in the sample; (S2a) comparing with a set threshold absolute amount; wherein the threshold absolute amount can distinguish between Sjögren's syndrome and systemic lupus erythematosus; optionally, the ZCCHC2 expression level in the Sjögren's syndrome patient sample is higher than the ZCCHC2 expression level in the systemic lupus erythematosus patient sample; or; (S2b) The expression level of ZCCHC2 in the sample was used as the input value in the algorithm for distinguishing Sjögren's syndrome from systemic lupus erythematosus; Preferably, the method further comprises the step of obtaining the expression levels of other markers in the subject sample, wherein the other markers include one or more of HES4, EIF2AK2, TTC21A, OTOF and ANKRD22.

7. A method for evaluating or screening diagnostic markers for Sjögren's syndrome, wherein: The method comprises the step of performing correlation or consistency analysis between potential biomarkers and ZCCHC2 expression levels.

8. A method for screening a therapeutic agent for Sjögren's syndrome, wherein: The method comprises the step of evaluating the expression level of ZCCHC2 in a sample treated with a therapeutic agent.

9. A marker for diagnosing Sjögren's syndrome or distinguishing Sjögren's syndrome from systemic lupus erythematosus, wherein: The marker comprises ZCCHC2 protein or a nucleic acid encoding ZCCHC2 protein; preferably, the marker further comprises one or more of HES4, EIF2AK2, TTC21A, OTOF and ANKRD22 proteins, or one or more of nucleic acids encoding HES4, EIF2AK2, TTC21A, OTOF and ANKRD22 proteins.

10. A method for treating Sjögren's syndrome, wherein: The method comprises the following steps: (1) Obtaining the ZCCHC2 expression level in the subject sample and comparing it with the ZCCHC2 expression level in the control sample; (2) If the ZCCHC2 expression level in the subject's sample is compared with the set threshold absolute amount, the subject is given glucocorticoids, antimalarial drugs, immunosuppressants, immunoglobulins, biologics, or nonsteroidal anti-inflammatory drugs.

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