Cerebrospinal fluid diagnostic markers for central nervous system lupus and their applications

Abstract

The present invention discloses cerebrospinal fluid markers for diagnosing central neuropsychiatric systemic lupus erythematosus, belonging to the field of biomedicine. The present invention provides the use of a reagent for quantitatively detecting markers in a sample in the preparation of a diagnostic kit for central neuropsychiatric systemic lupus erythematosus, and the markers are selected from one or more of MATN3, MERTK, VSIG4, and SERPINA3. The above-mentioned markers are related to patients with central neuropsychiatric systemic lupus erythematosus. Therefore, by quantitatively detecting the above-mentioned markers, it can be determined whether a subject has central neuropsychiatric systemic lupus erythematosus, and it can efficiently distinguish patients with central neuropsychiatric systemic lupus erythematosus from patients with central nervous system infections, non-infectious neurological diseases, and headache patients without any disease indications, greatly improving the probability of early and definite diagnosis of central neuropsychiatric systemic lupus erythematosus, and having important clinical significance.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to cerebrospinal fluid diagnostic markers for central nervous system lupus and their applications. Background Art

[0002] Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that mainly affects women of childbearing age. Among its complications, central nervous system lupus erythematosus (cNPSLE) is one of the most severe complications with a high fatality rate. The pathogenesis of cNPSLE is complex and unclear. Attacks by autoantibodies and inflammatory mediators, vascular lesions, disruption of the blood-brain barrier, and activation of the brain's innate immunity may all be involved in the development of cNPSLE. The manifestations of cNPSLE are diverse. Diffuse cNPSLE includes acute confusion, anxiety disorders, cognitive dysfunction, mood disorders, and psychosis; focal cNPSLE includes aseptic meningitis, cerebrovascular diseases, demyelinating syndromes, headache (including migraine and benign intracranial hypertension), movement disorders (such as chorea), myelopathy, and seizures. Among them, cognitive dysfunction, headache, acute confusion, cerebrovascular diseases, and epilepsy are the most common manifestations.

[0003] The manifestations of cNPSLE are complex and it is difficult to perform a pathological biopsy. Moreover, imaging examinations during the onset of the disease in patients are not only lacking in specificity but also difficult to cooperate with. So far, the diagnosis of cNPSLE remains a huge challenge. The main difficulty lies in how to rule out the possibility of infection when SLE patients present with neuropsychiatric symptoms, because the symptoms of cNPSLE and central infection may be completely similar, and it is difficult to distinguish them by routine cerebrospinal fluid tests, while the treatment directions of the two are completely opposite.

[0004] Cerebrospinal fluid (CSF) provides a direct detection window for monitoring changes in the central nervous system (CNS) and is the most valuable body fluid for assisting in the diagnosis of cNPSLE. However, there are few studies on cNPSLE CSF markers at present. Moreover, routine CSF tests cannot meet the diagnostic and differential diagnostic requirements of cNPSLE; the detection methods of chips or mass spectrometry are relatively complex. Therefore, the emergence of specific diagnostic markers is urgently needed. Summary of the Invention

[0005] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide cerebrospinal fluid diagnostic markers for central nervous system lupus and their applications. The present invention screens markers through high-throughput CSF proteomics and conducts large cohort verification, and screens out a series of CSF markers for diagnosing central nervous system lupus alone or in combination, which is expected to fill the gap in differential diagnostic markers between cNPSLE and CNS infection, improve the accuracy of clinical diagnosis, and thus contribute to achieving precise treatment and reducing the disability rate and mortality rate of this disease.

[0006] To achieve the above-mentioned invention objectives, the present invention provides the following technical solutions.

[0007] The present invention discloses the application of a reagent for quantitatively detecting a biomarker in a sample in the preparation of a diagnostic product for central neuropsychiatric systemic lupus erythematosus, characterized in that the biomarker is a combination of one or more of MATN3, MERTK, VSIG4, and SERPINA3.

[0008] Further, the product is used to distinguish central neuropsychiatric systemic lupus erythematosus from central nervous system infections, non-infectious central nervous system diseases, and headaches without any disease indications.

[0009] Further, the biomarker SERPINA3 and / or VSIG4 is / are used alone or in combination to distinguish between patients with central neuropsychiatric systemic lupus erythematosus and non-infectious central nervous system diseases.

[0010] Further, the biomarkers MATN3, MERTK, and VSIG4 are used alone or in combination to distinguish between patients with central neuropsychiatric systemic lupus erythematosus and central nervous system infections.

[0011] Further, the sample is a cerebrospinal fluid sample.

[0012] Further, the product is quantitatively detected by at least one of mass spectrometry, Western blotting, immunoprecipitation, and immunosorption.

[0013] The present invention also discloses a diagnostic kit for central neuropsychiatric systemic lupus erythematosus, characterized in that the biomarker is a combination of one or more of MATN3, MERTK, VSIG4, and SERPINA3.

[0014] Further, the biomarker SERPINA3 and / or VSIG4 is / are used alone or in combination to distinguish between patients with central neuropsychiatric systemic lupus erythematosus and non-infectious central nervous system diseases; the biomarkers MATN3, MERTK, and VSIG4 are used alone or in combination to distinguish between patients with central neuropsychiatric systemic lupus erythematosus and central nervous system infections.

[0015] Further, the kit is used to distinguish central neuropsychiatric systemic lupus erythematosus from central nervous system infections, non-infectious central nervous system diseases, and headaches without any disease indications.

[0016] Further, for the kit described in any of the above, it is characterized in that the kit further includes at least one of an immunosorption reagent and a mass spectrometry identification reagent.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0018] (1) The present invention provides diagnostic markers for the individual or combined use of VSIG4 and SERPINA3 for differential diagnosis of cNPSLE and controls, with strong specificity and high sensitivity, which helps to improve the accuracy of cNPSLE diagnosis; the controls are non-disease controls and patients with other non-infectious CSN diseases.

[0019] (2) The present invention provides diagnostic markers for the individual or combined use of MATN3, MERTK and VSIG4 for differential diagnosis of cNPSLE and CNS infections, with strong specificity and high sensitivity, filling the gap in the differential diagnosis of cNPSLE and CNS infections in clinical practice, helping to improve the prognosis and survival rate of patients, and helping to establish a new clinical diagnosis standard for cNPSLE.

[0020] (3) The diagnostic index provided by the present invention is the protein in cerebrospinal fluid. Cerebrospinal fluid provides a direct window for monitoring CNS changes and is the most valuable sample for the differential diagnosis of cNPSLE. Description of the Drawings

[0021] Figure 1 It is the volcano plot result of the comparison among the HC, NC, cNIC, and cNPSLE groups in the cerebrospinal fluid high-throughput mass spectrometry proteomics sequencing results.

[0022] Figure 2 It is the comparison of the relative expression levels of SERPINA3 and VSIG4 in the cerebrospinal fluid of cNPSLE, HC, and NC patients in the high-throughput mass spectrometry cohort. Among them, A is the comparison of the relative expression levels of SERPINA3 in the cerebrospinal fluid of cNPSLE, HC, and NC patients in the high-throughput mass spectrometry cohort; B is the comparison of the relative expression levels of VSIG4 in the cerebrospinal fluid of cNPSLE, HC, and NC patients in the high-throughput mass spectrometry cohort.

[0023] Figure 3 It is the comparison of the relative expression levels of MATN3, MERTK and VSIG4 in the cerebrospinal fluid of cNPSLE and cNIC patients in the high-throughput mass spectrometry cohort. Among them, A is the comparison of the relative expression levels of MATN3 in the cerebrospinal fluid of cNPSLE and cNIC patients in the high-throughput mass spectrometry cohort; B is the comparison of the relative expression levels of MERTK in the cerebrospinal fluid of cNPSLE and cNIC patients in the high-throughput mass spectrometry cohort; C is the comparison of the relative expression levels of VSIG4 in the cerebrospinal fluid of cNPSLE and cNIC patients in the high-throughput mass spectrometry cohort.

[0024] Figure 4Comparison of the expression levels of SERPINA3 and VSIG4 in the cerebrospinal fluid of cNPSLE and NC patients in Example 2. Among them, A is the comparison of the expression level of SERPINA3 in the cerebrospinal fluid of cNPSLE and NC patients in Example 2; B is the comparison of the expression level of VSIG4 in the cerebrospinal fluid of cNPSLE and NC patients in Example 2.

[0025] Figure 5 Comparison of the expression levels of MATN3, MERTK and VSIG4 in the cerebrospinal fluid of cNPSLE and cNIC patients in Example 2. Among them, A is the comparison of the expression level of MATN3 in the cerebrospinal fluid of cNPSLE and cNIC patients in Example 2; B is the comparison of the expression level of MERTK in the cerebrospinal fluid of cNPSLE and cNIC patients in Example 2; C is the comparison of the expression level of VSIG4 in the cerebrospinal fluid of cNPSLE and cNIC patients in Example 2.

[0026] Figure 6 ROC curves of SERPINA3 and VSIG4 for differentiating NC and cNPSLE patients in Example 2. Among them, A is the ROC curve of SERPINA3 and VSIG4 for differentiating NC and cNPSLE patients in Example 2 respectively; B is the ROC curve of SERPINA3 and VSIG4 combined for differentiating NC and cNPSLE patients in Example 2.

[0027] Figure 7 ROC curves of MATN3, MERTK and VSIG4 for differentiating cNIC and cNPSLE patients in Example 2. Among them, A is the ROC curve of MATN3, MERTK and VSIG4 for differentiating cNIC and cNPSLE patients in Example 2 respectively; B is the ROC curve of MATN3, MERTK and VSIG4 combined for differentiating cNIC and cNPSLE patients in Example 2.

[0028] Figure 8 Comparison of the expression levels of SERPINA3 and VSIG4 in the cerebrospinal fluid of cNPSLE and NC patients in Example 3. Among them, A is the comparison of the expression level of SERPINA3 in the cerebrospinal fluid of cNPSLE and NC patients in Example 3; B is the comparison of the expression level of VSIG4 in the cerebrospinal fluid of cNPSLE and NC patients in Example 3.

[0029] Figure 9Comparison of the expression levels of MATN3, MERTK, and VSIG4 in the cerebrospinal fluid of cNPSLE and cNIC patients in Example 3. Among them, A is the comparison of the expression levels of MATN3 in the cerebrospinal fluid of cNPSLE and cNIC patients in Example 3; B is the comparison of the expression levels of MERTK in the cerebrospinal fluid of cNPSLE and cNIC patients in Example 3; C is the comparison of the expression levels of VSIG4 in the cerebrospinal fluid of cNPSLE and cNIC patients in Example 3.

[0030] Figure 10 ROC curves of SERPINA3 and VSIG4 for differentiating NC and cNPSLE patients in Example 3. Among them, A is the ROC curve of SERPINA3 and VSIG4 for differentiating NC and cNPSLE patients in Example 3 respectively; B is the ROC curve of SERPINA3 and VSIG4 combined for differentiating NC and cNPSLE patients in Example 3.

[0031] Figure 11 ROC curves of MATN3, MERTK, and VSIG4 for differentiating cNIC and cNPSLE patients in Example 3. Among them, A is the ROC curve of MATN3, MERTK, and VSIG4 for differentiating cNIC and cNPSLE patients in Example 3 respectively; B is the ROC curve of MATN3, MERTK, and VSIG4 combined for differentiating cNIC and cNPSLE patients in Example 3. Detailed implementation manners

[0032] The present invention will be further described in detail below with specific examples. However, it should not be understood that the scope of the above-mentioned subject matter of the present invention is limited to the following examples, and all technologies implemented based on the content of the present invention belong to the scope of the present invention.

[0033] Unless otherwise specified, the reagents and materials used in the present invention are all commercially available.

[0034] Example 1. Biomarker screening.

[0035] Forty-nine clinical cerebrospinal fluid (CSF) samples were collected, including 20 patients with cNPSLE, 8 patients with CNS infection (cNIC), 10 patients with non-infectious CNS diseases (NC), and 11 healthy controls (HC). The healthy controls were headache patients without any disease indicators. All cNPSLE patients met the 2019 EULAR / ACR classification criteria for SLE. In 1999, the ACR proposed 12 manifestations of cNPSLE, which were divided into two major categories: diffuse and focal. The diffuse category included acute confusional state, cognitive dysfunction, psychosis, anxiety disorder, and mood disorder; the focal category included cerebrovascular disease, epilepsy, demyelinating syndrome, myelopathy, aseptic meningitis, headache (including migraine and benign intracranial hypertension), and movement disorders (such as chorea). The cNPSLE patients included in the study did not include patients with headache and mild cognitive dysfunction.

[0036] II. Experimental protocol.

[0037] (1) Samples were prepared using the filter-aided sample preparation (FASP) protocol.

[0038] (2) Peptide labeling was performed using the TMT reagent from Thermo Fisher Scientific for relative quantification.

[0039] (3) Proteomic analysis was performed on the prepared samples using the 120-min data-independent acquisition (DIA) method. This analysis was carried out in combination with the Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) and the EASY-nano-LC 1200 system.

[0040] (4) The raw data obtained was exported using Spectronaut for subsequent analysis.

[0041] III. Experimental results.

[0042] A total of 2,175 proteins were identified in the CSF samples by DIA-MS. As Figure 1 shown, in the CSF of cNPSLE patients, 242 proteins showed significant differences compared with HC (|log2(FC)| > 1 and adjusted p-value (P.adj) < 0.05), 45 proteins showed significant differences compared with cNIC, and 82 proteins showed significant differences compared with NC. Compared with HC, there were 107 differentially expressed proteins (DEPs) in the CSF of cNIC patients and 88 DEPs in NC patients.

[0043] Screening of cNPSLE-specific auxiliary diagnostic markers: After performing WGCNA analysis on the DIA-MS data of all patients in the cohort and taking the intersection with 242 DEPs comparing cNPSLE with HC, protein-protein interaction and Hub protein analysis were performed on the obtained 79 DEPs, resulting in 18 HubDEPs. Finally, through literature retrieval, two upregulated DEPs, SERPINA3 and VSIG4, were manually selected as candidate markers, and their relative expression levels in the cNPSLE, HC, and NC groups are as Figure 2 shown (**p < 0.01, ***p < 0.001).

[0044] Screening of cNPSLE-specific diagnostic markers to help exclude nerve infections: First, exclude the DEPs comparing cNIC with HC from the 242 DEPs comparing cNPSLE with HC, resulting in 192 DEPs with specific elevation in the CSF of cNPSLE patients; further, take the intersection of the 192 DEPs with the DEPs comparing cNPSLE and cNIC, obtaining 19 candidate proteins that are both cNPSLE disease-specific and have significant differences in expression levels compared with cNIC. Among them, three upregulated DEPs, MATN3, MERTK, and VSIG4, were used as candidate markers, and their relative expression levels in the cNPSLE and cNIC groups are as Figure 3 shown (**p < 0.01, ***p < 0.001).

[0045] Example 2.

[0046] I. Experimental samples.

[0047] Collect 115 clinical cerebrospinal fluid samples independent of the cohort in the screening stage, including 46 cNPSLE patients, 40 NC patients, and 29 cNIC patients. All cNPSLE patients meet the 2019 EULAR / ACR classification criteria for SLE. The cNPSLE patients included in the study do not include patients with headache and mild cognitive impairment.

[0048] II. Experimental protocol.

[0049] (1) Add the appropriately diluted sample of the standard product to the enzyme-linked immunosorbent assay (ELISA) plate coated with the capture antibody, seal the plate with a sealing film, and incubate at 25 - 37 °C for 1 - 2.5 h.

[0050] (2) Discard the liquid, add the biotin-labeled antibody working solution, seal the plate with a sealing film, and incubate at 25 - 37 °C for 1 h.

[0051] (3) Discard the liquid, wash, add the horseradish peroxidase-labeled streptavidin working solution, seal the plate with a sealing film, and incubate at 25 - 37 °C for 30 - 4 min.

[0052] (4) Discard the liquid, wash, add the chromogenic solution, incubate at 25 - 37 °C for 20 - 30 min, then add the stop solution, and read the absorbance in the microplate reader within 15 min.

[0053] (5) Result analysis: The data were analyzed using GraphPad Prism 9.0. One-way Anova and Dunnett correction were used for multiple comparisons, or the Mann–Whitney U test was used for two-group comparisons. The sensitivity and specificity of potential biomarker diagnosis were evaluated based on the receiver operating characteristic curve (ROC curve) and the area under the curve (AUC).

[0054] III. Experimental results.

[0055] (1) Compared with the NC group, both SERPINA3 and VSIG4 were significantly increased in cNPSLE patients, as Figure 4 shown; compared with the cNIC group, MATN3, MERTK, and VSIG4 were significantly increased in cNPSLE patients, as Figure 5 shown, (***p < 0.001).

[0056] (2) The two potential markers, SERPINA3 and VSIG4, alone or in combination, had diagnostic value in differentiating cNPSLE patients from the NC control group.

[0057] The ROC curves of SERPINA3 and VSIG4 for diagnosing cNPSLE are as Figure 6 shown in Figure A, and the AUCs were 0.885 (p < 0.0001, 95% CI 0.815 - 0.955) and 0.844 (p < 0.0001, 95% CI 0.760 - 0.928), respectively.

[0058] The ROC curve of SERPINA3 and VSIG4 combined for diagnosing cNPSLE is as Figure 6 shown in Figure B, and the AUC was 0.951 (p < 0.0001, 95% CI 0.901 - 0.992).

[0059] (3) The three potential markers, MATN3, MERTK, and VSIG4, alone or in combination, had diagnostic value in differentiating cNPSLE patients from the cNIC control group.

[0060] The ROC curves of MATN3, MERTK, and VSIG4 for diagnosing cNPSLE are as Figure 7As shown in Figure A, the AUCs of the three markers were 0.875 (p < 0.0001, 95% CI 0.794 - 0.956), 0.801 (p < 0.0001, 95% CI 0.697 - 0.906), and 0.724 (p < 0.01, 95% CI 0.602 - 0.846), respectively.

[0061] The ROC curve of MATN3, MERTK, and VSIG4 in the combined diagnosis of cNPSLE is as Figure 7 shown in Figure B, with an AUC of 0.921 (p < 0.0001, 95% CI 0.854 - 0.988).

[0062] Example 3.

[0063] I. Experimental samples.

[0064] Collect 38 clinical cerebrospinal fluid samples from another independent cohort, including 15 cNPSLE patients, 10 NC controls, and 13 cNIC controls. All cNPSLE patients met the 2019 EULAR / ACR classification criteria for SLE. The cNPSLE patients included in the study did not include patients with headache and mild cognitive impairment.

[0065] II. The experimental protocol is the same as that in Example 1.

[0066] III. Experimental results.

[0067] (1) Compared with the NC group, both SERPINA3 and VSIG4 were significantly increased in cNPSLE patients, as Figure 8 shown; compared with the cNIC group, MATN3, MERTK, and VSIG4 were all significantly increased in cNPSLE patients, as Figure 9 shown. (**p < 0.01).

[0068] (2) Two potential markers, SERPINA3 and VSIG4, alone or in combination, have diagnostic value in differentiating cNPSLE patients from NC controls.

[0069] The ROC curve of SERPINA3 and VSIG4 in the diagnosis of cNPSLE is as Figure 10 shown in Figure A, and the AUCs of the two markers were 0.873 (p < 0.01, 95% CI 0.739 - 1) and 0.807 (p < 0.05, 95% CI 0.621 - 0.992), respectively.

[0070] The ROC curve of SERPINA3 and VSIG4 in the combined diagnosis of cNPSLE is as Figure 10 shown in Figure B, with an AUC of 0.980 (p < 0.05, 95% CI 0.936 - 1).

[0071] (3) The three potential markers MATN3, MERTK, and VSIG4, either alone or in combination, have diagnostic value in differentiating cNPSLE patients from the cNIC control group.

[0072] The ROC curves of MATN3, MERTK, and VSIG4 for diagnosing cNPSLE are as Figure 11 shown in Figure A. The AUCs of the three markers are 0.785 (p < 0.05, 95% CI 0.598 - 0.972), 0.818 (p < 0.001, 95% CI 0.664 - 0.972), and 0.785 (p < 0.05, 95% CI 0.617 - 0.952), respectively.

[0073] The ROC curve of the combined diagnosis of cNPSLE by MATN3, MERTK, and VSIG4 is as Figure 11 shown in Figure B, and the AUC is 0.908 (p < 0.05, 95% CI 0.792 - 1).

[0074] In summary, the present invention provides the application of a series of CSF diagnostic markers in the preparation of cNPSLE CSF auxiliary diagnostic reagents. It is expected to fill the gap in the differential diagnostic markers between cNPSLE and neuroinfection, improve the accuracy of clinical diagnosis, thereby contributing to achieving precise treatment and reducing the disability and mortality rates of this disease.

[0075] The above are only the preferred embodiments of the present invention and are not used to limit the patent scope of the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of a reagent for quantitatively detecting markers in cerebrospinal fluid samples in the preparation of a diagnostic product for central nervous system psychotic systemic lupus erythematosus, wherein the marker is one or a combination of MATN3, MERTK, VSIG4 and SERPINA3; the product is used to distinguish central nervous system psychotic systemic lupus erythematosus from central nervous system infection or central nervous system psychotic systemic lupus erythematosus from non-infectious central nervous system diseases; the markers SERPINA3 and VSIG4 are used alone or in combination to distinguish between patients with central nervous system psychotic systemic lupus erythematosus and non-infectious central nervous system diseases; the markers MATN3, MERTK and VSIG4 are used alone or in combination to distinguish between patients with central nervous system psychotic systemic lupus erythematosus and central nervous system infection.

2. The use according to claim 1, characterized in that: The product is quantitatively detected by at least one of mass spectrometry, protein blotting, immunoprecipitation, and immunoadsorption.

3. The use according to claim 1, characterized in that: The product is a test kit.

4. A diagnostic kit for central nervous system psychiatric systemic lupus erythematosus, characterized in that: The kit is used to distinguish central nervous system psychotic systemic lupus erythematosus from central nervous system infection or central nervous system psychotic systemic lupus erythematosus from non-infectious central nervous system diseases; the kit contains reagents for detecting markers in cerebrospinal fluid samples, and the markers are a combination of SERPINA3 and VSIG4 or a combination of MATN3, MERTK and VSIG4; the markers SERPINA3 and VSIG4 are used in combination to distinguish central nervous system psychotic systemic lupus erythematosus from non-infectious central nervous system diseases; the markers MATN3, MERTK and VSIG4 are used in combination to distinguish central nervous system psychotic systemic lupus erythematosus from central nervous system infection.

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