Application of cerebrospinal fluid auxiliary diagnostic markers for neuropsychiatric lupus

Abstract

The application discloses a cerebrospinal fluid auxiliary diagnosis marker for neuropsychiatric lupus, and relates to the application of any one or more of TCN2, CST6, Trappin-2 and L-selectin as a diagnosis marker in the preparation of a cerebrospinal fluid auxiliary diagnosis reagent for neuropsychiatric lupus. The application provides four biomarkers of TCN2, etc. with high specificity and high sensitivity for diagnosing neuropsychiatric lupus, fills the blank in the field, further improves the accuracy of diagnosis, helps to realize timely early warning in clinic, and helps to establish a new clinical diagnosis standard for lupus encephalopathy.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine detection, and relates to application of a cerebrospinal fluid auxiliary diagnosis marker for neuropsychiatric systemic lupus erythematosus. BACKGROUND

[0002] Systemic lupus erythematosus (SLE) is a multi-organ involvement autoimmune disease mainly occurring in young women, which is characterized by abnormal immune tolerance leading to the production of anti-nuclear antibodies, anti-dsDNA and other pathogenic autoantibodies in the body, the formation of immune complex deposition, and the occurrence of multi-organ and multi-system involvement. Neuropsychiatric systemic lupus erythematosus (NPSLE) is one of the common complications of SLE, which mainly involves diffuse or focal damage of central nervous system and peripheral nervous system [1] . However, not all systemic lupus erythematosus will develop into neuropsychiatric systemic lupus erythematosus. The morbidity and mortality of NPSLE are only second to lupus nephritis, and the clinical manifestations are complex and variable. With the continuous in-depth research, the American College of Rheumatology (ACR) officially determined the standard naming and classification criteria of NPSLE in 1999, and defined 19 kinds of NPSLE symptoms, i.e. 12 kinds of central nervous system symptoms (epilepsy, aseptic meningitis, cerebrovascular disease, demyelination syndrome, etc.) and 7 kinds of peripheral nervous system symptoms (autonomic nervous disorder, cranial nerve disease, myasthenia gravis, etc.) [2] .

[0003] Due to the complex and variable clinical manifestations of NPSLE, the diagnosis techniques based on ACR criteria are constantly updated [3] , mainly including the following: ①serological and cerebrospinal fluid detection, including detection of autoantibodies and inflammatory factors. The autoantibody detection commonly used for the evaluation of NPSLE includes anti-phospholipid antibody, anti-ribosomal P antibody, anti-glutamate receptor antibody and anti-double-stranded DNA antibody [4] , for example, 70% of NPSLE patients will have high titer positive serum anti-double-stranded DNA antibody; anti-phospholipid antibodies include anti-cardiolipin antibody, anti-β2-glycoprotein 1 antibody and lupus anticoagulant, and non-specific headache, ischemic stroke and epilepsy are more likely to occur in NPSLE patients carrying high concentration of anti-phospholipid antibodies [7]; ② Brain imaging, a traditional non-invasive tool, can locate intracranial abnormalities in patients. The European League Against Rheumatism recommends MRI as the preferred examination for assessing intracranial and spinal lesions. It can also rule out other factors such as abscesses, infectious meningitis, and fungal aneurysms. With the continuous advancement of technology, more advanced imaging techniques have emerged, such as single photon emission computed tomography (SPECT) and positron emission computed tomography (PET / CT) [8] ; ③ Electrophysiological studies, including electroencephalogram, electromyogram, and nerve electrophysiology, are mainly used to detect seizures. Some studies have used electrophysiological techniques to detect neurological disorders in 1533 SLE patients, of which 207 had neurological disorders, including 70% with axonal neuropathy and 20% with demyelination

[12] . In addition to the above three types, there are also neuropsychological assessments for cognitive impairment and auxiliary examinations such as ultrasound.

[0004] The current clinical diagnosis procedure is as follows: after the patient presents with neuropsychiatric symptoms, drug-induced, infection-induced, and other disease-induced neuropsychiatric abnormalities are ruled out, serology, cerebrospinal fluid detection, MRI or CT examination, and neuropsychological testing are used to help diagnose NPSLE. In addition to presenting with NPSLE symptoms, patients must also meet three or more ACR criteria for SLE to be diagnosed with NPSLE. However, the ACR classification criteria have not been proven to be related to clinical diagnosis. For example, Karassa et al. found that anti-ribosomal P antibodies are not specific for the diagnosis of NPSLE and cannot distinguish between different symptom phenotypes

[13] ; The problem with brain imaging technology is that non-specific changes in brain structure are poorly correlated with the incidence of NPSLE, and imaging results depend on the judgment of experienced doctors. Therefore, due to the lack of specific non-invasive diagnostic markers, based on different diagnostic criteria and diverse disease manifestations, the incidence of NPSLE varies greatly among different population types and races (12%-95%), and early diagnosis of NPSLE has become a clinical challenge and focus, resulting in the diagnosis of NPSLE often occurring during the active phase of the disease, when the patient's brain tissue has often already been irreversibly damaged

[14] . Therefore, finding specific and highly sensitive biomarkers is crucial for clinical diagnosis and helps establish a reliable, simple, and relatively inexpensive diagnostic system.

[0005] Currently, there is little research on biomarkers for neuropsychiatric lupus. Kamala et al. used a chip based on aptamer binding to sequence the cerebrospinal fluid of NPSLE patients and found that lipocalin-2, M-CSF, IgM, and C3 in the cerebrospinal fluid have the potential to become biomarkers for the clinical diagnosis of NPSLE

[16] A protein chip of 94 antigens was used to screen autoantibodies in serum of NPSLE patients, which confirmed the correlation of anti-heparan sulfate, anti-histone H2B autoantibodies with NPSLE incidence

[17] Li Zhan et al. found that 21 metabolites (L-asparagine, epinephrine, guanidino acetic acid, etc.) can be used as biomarkers for diagnosing neuropsychiatric lupus alone or in combination

[18] . SUMMARY

[0006] The present application aims at providing the application of a group of diagnostic markers in the preparation of cerebrospinal fluid auxiliary diagnostic reagents for neuropsychiatric lupus to overcome the above-mentioned deficiencies of the prior art.

[0007] Another object of the present application is to provide the application of the detection of the group of diagnostic markers in the preparation of cerebrospinal fluid auxiliary diagnostic reagents for neuropsychiatric lupus.

[0008] Still another object of the present application is to provide a cerebrospinal fluid auxiliary diagnostic kit for neuropsychiatric lupus,

[0009] The object of the present application can be achieved by the following technical solutions:

[0010] The application of any one or more of TCN2, CST6, Trappin-2, L-selectin as diagnostic markers in the preparation of cerebrospinal fluid auxiliary diagnostic reagents for neuropsychiatric lupus.

[0011] As a preferred embodiment of the present application, the application of any one or both of TCN2 and CST6 as diagnostic markers in the preparation of cerebrospinal fluid auxiliary diagnostic reagents for distinguishing systemic lupus erythematosus and neuropsychiatric lupus.

[0012] As another preferred embodiment of the present application, the application of any one or more of CST6, Trappin-2, L-selectin as diagnostic markers in the preparation of cerebrospinal fluid auxiliary diagnostic reagents for distinguishing controls and neuropsychiatric lupus; the controls are healthy people or patients with other diseases other than systemic lupus erythematosus and neuropsychiatric lupus.

[0013] As a preferred embodiment of the present application, the application of any one or more of CST6 and Trappin-2 as diagnostic markers in the preparation of cerebrospinal fluid auxiliary diagnostic reagents for distinguishing controls and neuropsychiatric lupus; the controls are healthy people or patients with other diseases other than systemic lupus erythematosus and neuropsychiatric lupus.

[0014] The application of reagents for detecting any one or more of TCN2, CST6, Trappin-2, L-selectin in the preparation of cerebrospinal fluid auxiliary diagnostic reagents for neuropsychiatric lupus.

[0015] As a preferred embodiment of the present application, the reagent for detecting TCN2, CST6, Trappin-2, L-selectin is selected from the reagents required for detecting TCN2, CST6, Trappin-2, L-selectin by high-throughput protein chip assay, including blocking solution, CST6, L-selectin, Trappin-2, TCN2 specific biotin-labeled antibody, fluorescent agent-streptavidin.

[0016] As a preferred embodiment of the present application, the reagent for detecting any one or two of TCN2 and CST6 is used in the preparation of cerebrospinal fluid auxiliary diagnostic reagent for distinguishing systemic lupus erythematosus and neuropsychiatric lupus.

[0017] As a preferred embodiment of the present application, any one or more of CST6, Trappin-2, L-selectin is used as a diagnostic marker in the preparation of cerebrospinal fluid auxiliary diagnostic reagent for distinguishing control and neuropsychiatric lupus; the control is a healthy person or a patient with other diseases other than systemic lupus erythematosus and neuropsychiatric lupus.

[0018] As a preferred embodiment of the present application, any one or more of CST6 and Trappin-2 is used as a diagnostic marker in the preparation of cerebrospinal fluid auxiliary diagnostic reagent for distinguishing control and neuropsychiatric lupus; the control is a healthy person or a patient with other diseases other than systemic lupus erythematosus and neuropsychiatric lupus.

[0019] The cerebrospinal fluid auxiliary diagnostic kit for neuropsychiatric lupus is characterized in that it contains reagents for detecting any one or more of TCN2, CST6, Trappin-2, L-selectin.

[0020] As a preferred embodiment of the present application, the kit contains reagents for detecting any one or two of TCN2 and CST6.

[0021] As a preferred embodiment of the present application, the kit contains reagents for detecting any one or more of CST6, Trappin-2, L-selectin.

[0022] As a preferred embodiment of the present application, the kit contains reagents for detecting any one or two of CST6 and Trappin-2.

[0023] Beneficial effects:

[0024] (1) At present, there are few studies on biomarkers of neuropsychiatric lupus, and the application provides four specific and high-sensitivity biomarkers such as TCN2 for diagnosing neuropsychiatric lupus, fills the gap in the field, further improves the accuracy of diagnosis, and helps to realize timely early warning in clinic, and helps to establish a new standard for clinical diagnosis of lupus encephalopathy.

[0025] (2) The diagnostic index provided by the application is a protein in cerebrospinal fluid, and cerebrospinal fluid can more directly reflect the pathological changes in the brain than other peripheral indicators. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Content change of CST6 in cerebrospinal fluid of control group, SLE patients and NPSLE patients;

[0027] Figure 2 Content change of Trappin-2 in cerebrospinal fluid of control group, SLE patients and NPSLE patients;

[0028] Figure 3 Content change of L-selectin in cerebrospinal fluid of control group, SLE patients and NPSLE patients;

[0029] Figure 4 Content change of TCN2 in cerebrospinal fluid of control group, SLE patients and NPSLE patients;

[0030] Figure 5 ROC curve of CST6 for distinguishing control group and NPSLE patients;

[0031] Figure 6 ROC curve of Trappin-2 for distinguishing control group and NPSLE patients;

[0032] Figure 7 ROC curve of L-selectin for distinguishing control group and NPSLE patients;

[0033] Figure 8 ROC curve of CST6 and Trappin-2 for distinguishing control group and NPSLE patients;

[0034] Figure 9 ROC curve of CST6, Trappin-2 and L-selectin for distinguishing control group and NPSLE patients;

[0035] Figure 10 ROC curve of TCN2 for distinguishing SLE patients and NPSLE patients;

[0036] Figure 11ROC curve for differentiating SLE patients from NPSLE patients for CST6;

[0037] Figure 12 ROC curve for differentiating SLE patients from NPSLE patients for TCN2 and CST6 combined. DETAILED DESCRIPTION

[0038] Example 1

[0039] I. Experimental samples

[0040] A total of 64 clinical cerebrospinal fluid samples were collected, including 37 NPSLE patients, 18 SLE patients, and 9 control group samples. The classification criteria are shown in Tables 1 and 2. 55 patients (37 NPSLE patients and 18 SLE patients) met the new classification criteria for SLE in 2019 EULAR / ACR (see Table 3). NPSLE has diverse clinical manifestations, and there is no unified diagnostic standard internationally. In 1999, the American College of Rheumatology (ACR) defined 19 common neuropsychiatric symptoms of SLE, and any SLE patient who met one of the 19 neuropsychiatric symptoms was diagnosed as NPSLE. Among them, 12 symptoms are related to the central nervous system, including aseptic meningitis, cerebrovascular disease, lupus headache, demyelination syndrome, movement disorder (chorea), myelopathy, seizure, acute confusion state, anxiety, cognitive dysfunction, affective disorder, and psychosis; 7 symptoms are related to the peripheral nervous system, including acute inflammatory demyelination (Guillain-Barre syndrome), autonomic neuropathy, myasthenia gravis, cranial neuropathy, plexus neuropathy, mononeuropathy, and multiple neuropathy. The NPSLE patients included in the study were all SLE patients with seizures.

[0041] Table 1 Diagnostic criteria for Behcet's disease

[0042]

[0043] Table 2 Diagnostic criteria for Sjogren's syndrome

[0044]

[0045] Table 3 Diagnostic criteria for systemic lupus erythematosus

[0046]

[0047] SLE and NPSLE patients underwent necessary examinations to determine the disease activity of the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), and SLEDAI > 6 points represented active disease. The clinical staff performed lumbar puncture on the patients to collect cerebrospinal fluid, and the cerebrospinal fluid samples were immediately divided into siliconized polypropylene tubes and quickly frozen on dry ice. All samples were stored at -80°C or liquid nitrogen before measurement.

[0048] II. Experimental Protocol

[0049] (1) Blocking and sample incubation:

[0050] A. Take the slide chip out of the box, after equilibration at room temperature for 30 minutes, open the package bag, remove the sealing strip, then place the chip in a vacuum dryer or dry at room temperature for 1.5 hours.

[0051] B. Add 100 μL of 1x blocking solution to each well of the chip, incubate on a shaker at room temperature for 1 hour, avoiding the generation of air bubbles;

[0052] C. Remove the blocking solution, add 60 μL of sample stock solution to each well, one sample per array, incubate at 4°C overnight;

[0053] D. Clean the slide using the Thermo Scientific Wellwash Versa chip washer, in two steps, first use 1x wash solution I, 250 μL per well, wash 10 times, each time shake for 10 seconds, select high intensity, dilute 20x wash solution I with deionized water. Then use 1x wash solution II channel, 250 μL per well, wash 6 times, each time shake for 1 second, select high intensity, dilute 20x wash solution II with deionized water.

[0054] (2) Specific biotin-labeled antibody incubation of CST6, L-selectin, Trappin-2 and TCN2: add 70 μL of biotin-labeled antibody (Biotin-Conjugated Anti-cytokines) per well, incubate at room temperature for 2 hours with shaking, and wash as above.

[0055] (3) Fluorescent agent-streptavidin incubation: add 70 μL of 1500-fold diluted fluorescent agent-streptavidin (Cy3 equivalent) per well, seal the slide with a sealing strip, then cover the slide with aluminum foil to avoid light, incubate at room temperature for 2 hours with shaking, and wash as above.

[0056] (4) Fluorescence detection: scan the signal using a laser scanner (InnoScan 300 Microarray Scanner), using the Cy3 or green channel (excitation frequency = 532 nm).

[0057] (5) Result analysis: Data were analyzed by SPSS25 software, and t test was used to compare the differences of potential biomarkers between disease group and control group. Logistic regression was also performed, and the regression fitting degree was tested by likelihood ratio test, and the regression parameter estimate was tested by non-parametric test method. 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).

[0058] III. Experimental results

[0059] (1) Compared with the control group, CST6, Trappin-2 and L-selectin were significantly increased in NPSLE patients; compared with SLE patients, TCN2 and CST6 were significantly increased in NPSLE patients, as shown in Figures 1-4 (*P<0.05; **P<0.01).

[0060] (2) The diagnostic value of four potential biomarkers TCN2, CST6, Trappin-2 and L-selectin for NPSLE patients in distinguishing control group or SLE group. The area under the ROC curve (AUC) has been widely recognized as an inherent accuracy index for evaluating the authenticity of diagnostic tests. The AUC of completely worthless diagnostic tests is 0.5, and the AUC of ideal diagnostic tests is 1. Generally, AUC between 0.7 and 0.9 has certain diagnostic value.

[0061] ①Distinguish NPSLE group from control group:

[0062] The ROC curves of CST6, Trappin-2 and L-selectin for diagnosing NSPLE are shown in Figures 5-7 , respectively, in which the AUCs can reach 0.778 (P=0.0104; 95% CI: 0.6357-0.9199), 0.793 (P=0.0069; 95% CI: 0.6366-0.9490), and 0.844 (P=0.0015; 95% CI: 0.7289-0.9588), respectively.

[0063] The ROC curve of CST6 and Trappin-2 for combined diagnosis of NPSLE is shown in Figure 8 , in which AUC=0.851 (P=0.0012; 95% CI: 0.7420-0.9607). In addition, any two of the three proteins combined to diagnose NPSLE also has good diagnostic value. The ROC curve of CST6, Trappin-2 and L-selectin for combined diagnosis of NPSLE is shown in Figure 9As shown, AUC = 0.884 (P = 0.0004; 95% CI: 0.7890-0.9797).

[0064] ② Distinguish between NPSLE group and SLE group:

[0065] The ROC curves for TCN2 and CST6 in diagnosing NPSLE are as follows: Figures 10-11 As shown, the AUCs were 0.703 (P = 0.0155; 95% CI: 0.5560-0.8494) and 0.739 (P = 0.0043; 95% CI: 0.6025-0.8750), respectively.

[0066] The ROC curve for the combined diagnosis of NPSLE using TCN2 and CST6 is as follows: Figure 12 As shown, AUC = 0.814 (P = 0.0002; 95% CI: 0.6821-0.9455).

[0067] References and patents:

[0068] [1] Vivaldo JF, de Amorim JC, Julio PR, et al. Definition of NPSLE: Does the ACR Nomenclature Still Hold? Front Med(Lausanne).2018May 31;5:138.

[0069] [2]The American College of Rheumatology nomenclature and casedefinitions for neuropsychiatric lupus syndromes.Arthritis Rheum.1999Apr;42(4):599-608.

[0070] [3]Hochberg MC.Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus.ArthritisRheum.1997Sep;40(9):1725.

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[0072] [5] Hanly JG, Robichaud J, Fisk JD. Anti-NR2 glutamate receptor antibodies and cognitive function in systemic lupus erythematosus. J Rheumatol. 2006 Aug; 33(8): 1553-8.

[0073] [6] Clark KE, Clark CN, Rahman A. A critical analysis of the tools to evaluate neuropsychiatric lupus. Lupus. 2017 Apr; 26(5): 504-509.

[0074] [7] Coín MA, Vilar-López R, Peralta-Ramírez I, et al. The role of antiphospholipid autoantibodies in the cognitive deficits of patients with systemic lupus erythematosus. Lupus. 2015 Jul; 24(8): 875-9.

[0075] [8] Cohen D, Rijnink EC, Nabuurs RJ, et al. Brain histopathology in patients with systemic lupus erythematosus: identification of lesions associated with clinical neuropsychiatric lupus syndromes and the role of complement. Rheumatology (Oxford). 2017 Jan;56(1):77-86.

[0076] [9] Mackay M, Tang CC, Vo A. Advanced neuroimaging in neuropsychiatric systemic lupus erythematosus. Curr Opin Neurol. 2020 Jun;33(3):353-361.

[0077]

[10] Curiel R, Akin EA, Beaulieu G, et al. PET / CT imaging in systemic lupus erythematosus. Ann N Y Acad Sci. 2011 Jun;1228:71-80.

[0078]

[11] Inglese F, Kim M, Steup-Beekman GM, et al. MRI-Based Classification of Neuropsychiatric Systemic Lupus Erythematosus Patients With Self-Supervised Contrastive Learning. Front Neurosci. 2022 Feb 16;16:695888.

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[12] Florica B, Aghdassi E, Su J, et al. Peripheral neuropathy in patients with systemic lupus erythematosus. Semin Arthritis Rheum. 2011 Oct;41(2):203-11.

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[13] Varley JA, Andersson M, Grant E, et al. Absence of Neuronal Autoantibodies in Neuropsychiatric Systemic Lupus Erythematosus. Ann Neurol. 2020 Dec;88(6): 1244-1250.

[14] Schwartz N, Stock AD, Putterman C. Neuropsychiatric lupus: new mechanistic insights and future treatment directions. Nat Rev Rheumatol. 2019 Mar;15(3): 137-152.

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[15] Zardi EM, Taccone A, Marigliano B, et al. Neuropsychiatric systemic lupus erythematosus: tools for the diagnosis. Autoimmun Rev. 2014 Aug;13(8):831-9.

[0082]

[16] Vanarsa K, Sasidharan P, Duran V, et al. Aptamer-based screen of Neuropsychiatric Lupus cerebrospinal fluid reveals potential biomarkers that overlap with the choroid plexus transcriptome. Arthritis Rheumatol. 2022 Jan 31.

[0083]

[17] van der Meulen PM, Barendregt AM, Cuadrado E, et al. Protein array autoantibody profiles to determine diagnostic markers for neuropsychiatric systemic lupus erythematosus. Rheumatology (Oxford). 2017 Aug 1 ;56(8): 1407-1416.

[0084]

[18] Li Z, Sun XL, Li X. A diagnostic marker for neuropsychiatric lupus and a detection kit thereof[P]. Beijing: CN111562390A, 2020-08-21.

Claims

1. The use of reagents for detecting any one of the following substances in the preparation of cerebrospinal fluid auxiliary diagnostic reagents for neuropsychiatric lupus: (A1) TCN2; (A2) TCN2 and any one or more of CST6, Trappin-2, L-selectin. The detection reagents are selected from high-throughput protein chip detection reagents for detecting the content of the substances, and the detection reagents include a blocking solution, biotin-labeled antibodies, and a fluorescent agent-streptavidin. The use of reagents for detecting TCN2 or TCN2 and CST6 in the preparation of cerebrospinal fluid auxiliary diagnostic reagents for distinguishing systemic lupus erythematosus and neuropsychiatric lupus.

2. Use according to claim 1, characterized in that, 4. The use of reagents for detecting TCN2 or TCN2 and CST6 in the preparation of cerebrospinal fluid auxiliary diagnostic reagents for distinguishing systemic lupus erythematosus and neuropsychiatric lupus.

3. Use according to claim 2, characterized in that ​ ​

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