Cerebrospinal fluid marker for diagnosing central neuropsychiatric systemic lupus erythematosus and use of marker

Abstract

The present invention relates to the field of biomedicine. Disclosed is a cerebrospinal fluid marker for diagnosing central neuropsychiatric systemic lupus erythematosus. The present invention provides a use of a reagent for quantitatively measuring a marker in a sample in the preparation of a diagnostic kit for central neuropsychiatric systemic lupus erythematosus. The marker is selected from one or more of MATN3, MERTK, VSIG4, and SERPINA3. The marker is associated with patients with central neuropsychiatric systemic lupus erythematosus. Therefore, a determination as to whether a subject suffers from central neuropsychiatric systemic lupus erythematosus can be made by quantitatively measuring the marker, so that patients with central neuropsychiatric systemic lupus erythematosus can be efficiently distinguished from patients with central nervous system infection, patients with non-infectious nervous system diseases, and headache patients without any disease indication, thereby greatly improving the probability of early and definite diagnosis of central neuropsychiatric systemic lupus erythematosus. The present invention has important clinical significance.

Description

Central nervous system neuropsychiatric lupus cerebrospinal fluid diagnostic marker and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a central nervous system neuropsychiatric lupus cerebrospinal fluid diagnostic marker and application thereof. BACKGROUND

[0002] Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that mainly affects women of childbearing age. Among its complications, central nervous system neuropsychiatric systemic lupus erythematosus (cNPSLE) is one of the most severe complications with high mortality. The pathogenesis of cNPSLE is complex and unclear, and the attack of autoantibodies and inflammatory mediators, vascular lesions, destruction of the blood-brain barrier, and activation of the brain's innate immunity may all be involved in the development of cNPSLE. cNPSLE has diverse manifestations, diffuse cNPSLE includes acute confusion, anxiety, cognitive impairment, mood disorders, and psychosis; focal cNPSLE includes aseptic meningitis, cerebrovascular disease, demyelination syndrome, headache (including migraine and benign intracranial hypertension), movement disorders (such as chorea), myelopathy, and seizures, among which cognitive impairment, headache, acute confusion, cerebrovascular disease, and seizures are the most common manifestations.

[0003] The manifestations of cNPSLE are complex and difficult to perform pathological biopsy, and imaging examination is not only lack of specificity but also difficult to complete when the patient is ill, and so far the diagnosis of cNPSLE remains a great challenge. The main difficulty lies in how to rule out infection when SLE patients present with neuropsychiatric symptoms, because the symptoms of cNPSLE and central nervous system infection may be completely similar, and routine cerebrospinal fluid detection is difficult to distinguish, while the treatment directions of the two are completely different.

[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, but there are few studies on CSF markers for cNPSLE. Moreover, routine CSF tests cannot meet the needs of diagnosis and differential diagnosis of cNPSLE; the detection methods of chips or mass spectrometry are relatively complex to operate. Therefore, the emergence of specific diagnostic markers is urgently needed. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application aims to provide a central nervous mental lupus cerebrospinal fluid diagnostic marker and application thereof. The present application screens a series of CSF markers for diagnosing central nervous mental lupus alone or in combination through high-throughput CSF proteomics and large cohort verification, so as to fill the gap in the differential diagnosis markers of cNPSLE and CNS infection, improve the accuracy of clinical diagnosis, and thus help to realize precise treatment and reduce the morbidity and mortality of the disease.

[0006] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions.

[0007] The application discloses application of a reagent for quantitatively detecting a marker in a sample in preparation of a diagnostic product of central nervous mental systemic lupus erythematosus, and has the characteristics that the marker is one or a combination of several of MATN3, MERTK, VSIG4 and SERPINA3.

[0008] Further, the product is used for distinguishing central nervous mental systemic lupus erythematosus from central nervous system infection, non-infectious central nervous system disease and headache without any disease indication.

[0009] Further, the marker SERPINA3 and VSIG4 are used alone or in combination to distinguish central nervous mental systemic lupus erythematosus from non-infectious central nervous system disease.

[0010] Further, the marker MATN3, MERTK and VSIG4 are used alone or in combination to distinguish central nervous mental systemic lupus erythematosus from central nervous system infection.

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

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

[0013] The application further discloses a diagnostic kit for central nervous mental systemic lupus erythematosus, and has the characteristics that the marker is one or a combination of several of MATN3, MERTK, VSIG4 and SERPINA3.

[0014] Further, the marker SERPINA3 and VSIG4 are used alone or in combination to distinguish central nervous mental systemic lupus erythematosus from non-infectious central nervous system disease; and the marker MATN3, MERTK and VSIG4 are used alone or in combination to distinguish central nervous mental systemic lupus erythematosus from central nervous system infection.

[0015] Further, the kit is used for distinguishing central nervous psychogenic systemic lupus erythematosus from central nervous system infection, non-infectious central nervous system diseases and headache without any disease indication.

[0016] Further, the kit of any one of the above has the feature that the kit further comprises at least one of an immunoadsorption reagent, a mass spectrometry identification reagent.

[0017] Compared with the prior art, the present application has the following beneficial effects.

[0018] (1) The present application provides a diagnostic marker of VSIG4 and SERPINA3 alone or in combination for differential diagnosis of cNPSLE and controls, which has high specificity and sensitivity and helps to improve the accuracy of cNPSLE diagnosis; the controls are non-disease controls and other non-infectious CSN disease patients.

[0019] (2) The present application provides a diagnostic marker of MATN3, MERTK and VSIG4 alone or in combination for differential diagnosis of cNPSLE and CNS infection, which has high specificity and sensitivity and fills the gap in the differential diagnosis of cNPSLE and CNS infection in clinic, helps to improve the prognosis and survival rate of patients and help to establish new standards for the clinical diagnosis of cNPSLE.

[0020] (3) The diagnostic index provided by the present application is a protein in cerebrospinal fluid, and cerebrospinal fluid provides a direct window for monitoring changes in CNS, which is the most valuable sample for diagnosing and differentiating cNPSLE. BRIEF DESCRIPTION OF DRAWINGS

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

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

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

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

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

[0026] Figure 6 is the ROC curve of SERPINA3 and VSIG4 distinguishing NC and cNPSLE patients in Example Two. Among them, A is the ROC curve of SERPINA3 and VSIG4 respectively distinguishing NC and cNPSLE patients in Example Two; B is the ROC curve of SERPINA3 and VSIG4 combined to distinguish NC and cNPSLE patients in Example Two.

[0027] Figure 7 is the ROC curve of MATN3, MERTK and VSIG4 distinguishing cNIC and cNPSLE patients in Example Two. Among them, A is the ROC curve of MATN3, MERTK and VSIG4 respectively distinguishing cNIC and cNPSLE patients in Example Two; B is the ROC curve of MATN3, MERTK and VSIG4 combined to distinguish cNIC and cNPSLE patients in Example Two.

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

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

[0030] Figure 10 is the ROC curve of SERPINA3 and VSIG4 in distinguishing NC and cNPSLE patients in Example Three. Wherein, A is the ROC curve of SERPINA3 and VSIG4 respectively in distinguishing NC and cNPSLE patients in Example Three; B is the ROC curve of SERPINA3 and VSIG4 combined in distinguishing NC and cNPSLE patients in Example Three.

[0031] Figure 11 is the ROC curve of MATN3, MERTK and VSIG4 in distinguishing cNIC and cNPSLE patients in Example Three. Wherein, A is the ROC curve of MATN3, MERTK and VSIG4 respectively in distinguishing cNIC and cNPSLE patients in Example Three; B is the ROC curve of MATN3, MERTK and VSIG4 combined in distinguishing cNIC and cNPSLE patients in Example Three. DETAILED DESCRIPTION

[0032] The application will be further described in the following specific examples. However, it should not be understood that the scope of the above-mentioned subject matter of the application is limited to the following examples, and any technology achieved based on the content of the application is within the scope of the application.

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

[0034] Example One, Marker Screening.

[0035] Forty-nine CSF samples were collected, including 20 cNPSLE patients, 8 CNS infection (cNIC) patients, 10 non-infectious CNS disease patients (NC), and 11 healthy controls (HC). The healthy controls were patients with headache without any disease indications. The cNPSLE patients met the 2019 EULAR / ACR classification criteria for SLE. The 1999 ACR proposed 12 manifestations of cNPSLE, which were divided into two categories: diffuse and focal. The diffuse manifestations included acute confusion, cognitive dysfunction, psychosis, anxiety, and mood disorders; the focal manifestations included cerebrovascular disease, epilepsy, demyelination 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 impairment.

[0036] II. Experimental protocol

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

[0038] (2) The TMT reagents from Thermo Fisher Scientific were used for peptide labeling to achieve relative quantification.

[0039] (3) The prepared samples were subjected to proteomic analysis using the 120 min DIA method. The analysis was performed in combination with an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) and an EASY-nano-LC 1200 system.

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

[0041] III. Experimental results

[0042] A total of 2175 proteins were identified in the CSF samples by DIA-MS. As shown in Figure 1, 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 the CSF of NC patients.

[0043] cNPSLE-specific auxiliary diagnostic marker screening: After WGCNA analysis of DIA-MS data of all patients in the cohort, the intersection of 242 DEPs of cNPSLE compared with HC was obtained, and protein interaction and Hub protein analysis was performed on the obtained 79 DEPs. 18 Hub DEPs were obtained, and finally SERPINA3 and VSIG4, two up-regulated DEPs, were manually screened as candidate markers according to literature retrieval. The relative expression of the two in the cNPSLE, HC and NC groups is shown in Figure 2 (**p<0.01, ***p<0.001).

[0044] cNPSLE-specific diagnostic marker screening to help rule out neuroinfection: First, the DEPs of cNIC compared with HC were excluded from the 242 DEPs of cNPSLE compared with HC, and 192 DEPs of cNPSLE patient CSF-specific increase were obtained. Further, the intersection of 192 DEPs and DEPs of cNPSLE and cNIC was obtained, and 19 candidate proteins with both cNPSLE disease specificity and significant differences in expression compared with cNIC were obtained. Among them, MATN3, MERTK and VSIG4, three up-regulated DEPs, were used as candidate markers. The relative expression of the three in the cNPSLE and cNIC groups is shown in Figure 3 (**p<0.01, ***p<0.001).

[0045] Example Two.

[0046] I. Experimental samples

[0047] 115 clinical cerebrospinal fluid samples were collected independently of the screening phase cohort, including 46 cNPSLE patients, 40 NC patients and 29 cNIC patients. The cNPSLE patients all 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.

[0048] II. Experimental scheme

[0049] (1) The sample was diluted with the standard and added to the enzyme-labeled plate coated with the capture antibody. After sealing the plate with the sealing film, it was incubated at 25-37°C for 1-2.5h.

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

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

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

[0053] (5) Result analysis: data were analyzed by GraphPad Prism 9.0, One-way Anova and Dunnett correction were used for multiple comparisons, or Mann-Whitney U test was used for two group comparisons. The sensitivity and specificity of potential biomarkers for diagnosis were evaluated according to 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, SERPINA3 and VSIG4 were significantly increased in cNPSLE patients, as shown in Figure 4; compared with the cNIC group, MATN3, MERTK, and VSIG4 were significantly increased in cNPSLE patients, as shown in Figure 5, (***p<0.001).

[0056] (2) Two potential markers SERPINA3 and VSIG4 alone or in combination had diagnostic value in distinguishing cNPSLE patients from NC controls.

[0057] The ROC curve of SERPINA3 and VSIG4 in each group for diagnosing cNPSLE is shown in Figure 6A, 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 in combination for diagnosing cNPSLE is shown in Figure 6B, and the AUC was 0.951 (p<0.0001, 95% CI 0.901-0.992).

[0059] (3) Three potential markers MATN3, MERTK, and VSIG4 alone or in combination had diagnostic value in distinguishing cNPSLE patients from cNIC controls.

[0060] The ROC curve of MATN3, MERTK, and VSIG4 in each group for diagnosing cNPSLE is shown in Figure 7A, and 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 combination for diagnosing cNPSLE is shown in FIG. 7B, and the AUC is 0.921 (p<0.0001, 95% CI 0.854-0.988).

[0062] Example Three.

[0063] I. Experimental samples

[0064] Another independent cohort of 38 clinical CSF samples was collected, including 15 cNPSLE patients, 10 NC controls, and 13 cNIC controls. The 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. Experimental scheme same as Example 1.

[0066] III. Experimental results

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

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

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

[0070] The ROC curve of SERPINA3 and VSIG4 in combination for diagnosing cNPSLE is shown in FIG. 10B, and the AUC is 0.980 (p<0.05, 95% CI 0.936-1).

[0071] (3) Three potential markers MATN3, MERTK, and VSIG4 alone or in combination have diagnostic value in distinguishing cNPSLE patients from cNIC controls.

[0072] The ROC curve of MATN3, MERTK and VSIG4 in diagnosing cNPSLE is shown in Figure 11A, and the AUC of the three markers is 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 MATN3, MERTK and VSIG4 in diagnosing cNPSLE is shown in Figure 11A, and the AUC of the three markers is 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.

[0074] In summary, the application provides a series of CSF diagnostic markers for preparing cNPSLE CSF auxiliary diagnostic reagents. It is expected to fill the gap in the differential diagnosis markers of cNPSLE and neuroinfection, improve the accuracy of clinical diagnosis, and help achieve precise treatment and reduce the morbidity and mortality of the disease.

[0075] The above only describes the preferred embodiments of the application and is not used to limit the patent scope of the application. For those skilled in the art, the application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the application shall be included in the protection scope of the application.

Claims

1. Use of reagents for quantitative detection of markers in cerebrospinal fluid samples, said markers being one or a combination of MATN3, MERTK, VSIG4 and SERPINA3, in the manufacture of a diagnostic product for central nervous psychiatric systemic lupus erythematosus, said product being used to distinguish central nervous psychiatric systemic lupus erythematosus from central nervous system infection or central nervous psychiatric systemic lupus erythematosus from non-infectious central nervous system disease; said markers SERPINA3 and VSIG4, alone or in combination, being used to distinguish central nervous psychiatric systemic lupus erythematosus from non-infectious central nervous system disease; said markers MATN3, MERTK and VSIG4, alone or in combination, being used to distinguish central nervous psychiatric systemic lupus erythematosus from central nervous system infection.

2. Use according to claim 1, characterized in that, The product is quantitatively detected by at least one of the following methods: mass spectrometry, Western blot, immunoprecipitation, immunoadsorption.

3. Use according to claim 1, characterized in that, The product is a kit.

4. A diagnostic kit for central nervous psychogenic systemic lupus erythematosus, characterized by, The kit is used to distinguish central nervous psychiatric systemic lupus erythematosus from central nervous system infection or central nervous psychiatric systemic lupus erythematosus from non-infectious central nervous system disease; said kit comprising reagents for detection of markers in cerebrospinal fluid samples, said markers being a combination of SERPINA3 and VSIG4 or a combination of MATN3, MERTK and VSIG4; said markers SERPINA3 and VSIG4, in combination, being used to distinguish central nervous psychiatric systemic lupus erythematosus from non-infectious central nervous system disease; said markers MATN3, MERTK and VSIG4, in combination, being used to distinguish central nervous psychiatric systemic lupus erythematosus from central nervous system infection.

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