Application of diagnostic marker for neuromyelitis optica pedigree diseases

Abstract

The invention provides application of a diagnostic marker for neuromyelitis optica pedigree diseases, and belongs to the technical field of biological detection. According to the invention, reticulocalbin-1 (RCN1) in a CREC family is obtained through screening, is closely related to NMOSD, can be used as a diagnostic marker for clinical diagnosis, and has the advantages of high specificity, simple and convenient diagnosis process and excellent diagnostic performance; according to the present invention, with the combination diagnosis of the RCN1, the RCN3 (Regulocalbin-3) and the SDF4 (45kDa calcium-binding protein), the diagnosis efficiency can be further improved;

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and in particular relates to the application of a diagnostic marker for neuromyelitis optica spectrum disorders. Background Art

[0002] The statements in this section merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Neuromyelitis optica spectrum disorders (NMOSD) are a group of autoimmune-mediated inflammatory demyelinating diseases of the central nervous system (CNS) that primarily affect the optic nerves and spinal cord. NMOSD is associated with a high relapse rate and morbidity, with approximately 60% of patients experiencing a relapse within 1 year and 90% within 3 years. Recurrent attacks can lead to severe consequences such as blindness, paralysis, and death, placing a significant economic burden on patients' families and society. NMOSD is most common in adults, with a prevalence of 0.37 to 0.40 cases per 100,000 people. It primarily affects individuals aged 30 to 40 years, with a higher prevalence in women than in men. NMOSD is primarily caused by abnormal immune function resulting from various causes, which in turn triggers an abnormal inflammatory response. Given the diverse range of neuroinflammatory diseases, differentiation based solely on inflammatory markers is difficult. Therefore, identifying biomarkers for the diagnosis and treatment of NMOSD is of great clinical significance.

[0004] It is currently believed that positive blood aquaporin-4 (AQP4) antibodies are associated with the pathogenesis of NMOSD. In 2015, an international NMOSD diagnostic expert group established universally accepted diagnostic criteria for NMOSD, which include six core clinical features: optic neuritis, acute myelitis, area postrema syndrome, acute brainstem syndrome, narcolepsy or acute diencephalic syndrome with NMOSD-typical magnetic resonance imaging lesions, and cerebral syndrome with NMOSD-typical white matter lesions. The current diagnostic criteria are categorized by serotype as AQP4 antibody-positive or AQP4 antibody-negative / unknown, along with six core clinical features. While positive AQP4 antibodies and one core clinical feature are sufficient for the diagnosis of NMOSD, some patients with negative / unknown AQP4 antibodies require magnetic resonance imaging (MRI) alone for diagnosis. Previous clinical imaging and laboratory studies have also found no significant differences in serum AQP4 antibody positivity among NMOSD patients: AQP4 IgG testing may be negative at some point during the course of the first episode of NMOSD. Therefore, there is a lack of sensitive and specific auxiliary diagnostic biomarkers for the early diagnosis of NMOSD, especially in cases with atypical clinical and imaging features, which can also distinguish other neurological diseases such as multiple sclerosis (MS), acute disseminated encephalomyelitis (ADEM), and encephalitis. Therefore, it is extremely important to find a solution for the early diagnosis of NMOSD and long-term prevention of relapses. Summary of the Invention

[0005] The present invention screened and obtained Reticulocalbin-1 (RCN1) in the CREC family, which is closely related to NMOSD and can be used as a diagnostic marker for clinical diagnosis. It has high specificity, a simple diagnostic process, and excellent diagnostic performance. The combined diagnosis of RCN1 with RCN3 (Reticulocalbin-3) and SDF4 (45kDa calcium-binding protein) can further improve diagnostic efficiency.

[0006] Specifically, the technical solutions of the present invention are as follows:

[0007] In a first aspect, the present invention provides a use of a reagent for detecting RCN1 and related biological materials in the preparation of a product for screening, diagnosing, detecting, monitoring NMOSD or predicting the progression of NMOSD.

[0008] Optionally, the RCN1 is derived from plasma or cerebrospinal fluid.

[0009] Optionally, the relevant biological material includes RCN1 mRNA.

[0010] Optionally, the RCN1 mRNA is derived from peripheral blood mononuclear cells (PBMC).

[0011] Optionally, the product also includes reagents for detecting RCN3 and SDF4.

[0012] Optionally, the RCN3 and SDF4 are derived from plasma.

[0013] The second aspect of the present invention provides a product for screening, diagnosing, detecting, monitoring NMOSD or predicting the progression of NMOSD, which comprises a substance for detecting the expression of RCN1 and related biological materials in a sample to be tested based on a high-throughput sequencing method and / or a quantitative PCR method and / or a probe hybridization method.

[0014] Optionally, the product includes reagents for detecting RCN3 and SDF4.

[0015] A third aspect of the present invention provides a system for screening, diagnosing, detecting, monitoring, or predicting the progression of NMOSD, comprising:

[0016] An analysis module, configured to determine the expression level of a detection substance selected from RCN1 and its related biological materials in a test sample of a subject;

[0017] The evaluation module is used to judge the condition of the subject according to the expression levels of RCN1 and its related biological materials determined by the analysis module.

[0018] Optionally, a combined detection module is also included, which is used to detect the expression levels of RCN3, SDF4 and related biological materials in the test sample of the subject.

[0019] One or more of the above technical solutions have the following beneficial effects:

[0020] This invention demonstrates for the first time that the CREC family, represented by RCN1, is associated with NMOSD and can serve as a biomarker for NMOSD. The findings are as follows: (1) RCN1 and its related biomaterials (mRNA) are upregulated in the plasma and peripheral blood mononuclear cells (PBMCs) of NMOSD patients, while downregulated in the cerebrospinal fluid (CSF); (2) The plasma RCN1 protein level in acute NMOSD patients is correlated with the severity of NMOSD, as shown by the higher the plasma RCN1 protein level, the older the patient's first onset age, the fewer the number of onsets, and the lower the EDSS (Disease Disability Score); (3) RCN1 protein expression levels are positively correlated with the age of first onset of NMOSD and can serve as an early screening marker; (4) Increased plasma RCN1 protein levels and RCN1 mRNA levels in PBMCs, combined with decreased cerebrospinal fluid RCN1 protein levels, can serve as diagnostic markers for NMOSD. (5) Combined detection of plasma RCN1 protein levels, plasma RCN3 protein levels, and plasma SDF4 protein levels as a combined diagnostic marker for NMOSD has higher sensitivity.

[0021] Based on this, the present invention provides a scheme for diagnosing NMOSD using this marker, which can achieve accurate diagnosis of NMOSD and differential diagnosis from MS compared with traditional NMOSD diagnosis, specifically including: (1) Compared with the normal population, the RCN1 protein content in the plasma of patients in the acute phase of NMOSD is significantly increased and significantly correlated with the onset of NMOSD; while the RCN1 protein content in the plasma of MS patients does not change much, so the detection of RCN1 protein content in plasma is used for early screening or diagnosis of NMOSD; (2) The detection of plasma RCN1 protein content combined with the RCN1 mRNA level in PBMC and the RCN1 protein content in cerebrospinal fluid can improve the diagnostic efficacy of NMOSD, which is different from that of patients with encephalitis; (3) The combined diagnosis of NMOSD by plasma RCN1 protein content, RCN3 protein content and SDF4 protein content can improve the diagnostic efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0023] Figure 1 This is a statistical graph of the secretion levels of RCN1 in the plasma of controls, acute NMOSD patients, MS patients, and encephalitis patients screened in the examples of the present invention.

[0024] Figure 2 This is a statistical graph showing the mRNA levels of CREC family members RCN1, RCN2, RCN3, SDF4 and CAUL in blood PBMCs of controls and acute-stage NMOSD patients screened in the examples of the present invention.

[0025] Figure 3 This is a statistical graph of the secretion levels of RCN1 in the cerebrospinal fluid (CSF) of controls (pressure hydrocephalus), acute NMOSD patients, and encephalitis patients in the screening set in the examples of the present invention.

[0026] Figure 4 Statistical graphs of the secretion levels of RCN1, RCN3, and SDF4 in the plasma of controls and acute-stage NMOSD patients, respectively, of the validation set in the embodiments of the present invention, wherein (A) is the statistical graph of RCN1, (B) is the statistical graph of RCN3, and (C) is the statistical graph of SDF4.

[0027] Figure 5 This is a statistical result diagram of the mRNA levels of CREC family members RCN1, RCN2, RCN3, SDF4 and CAUL in blood PBMCs of controls and acute-stage NMOSD patients in the validation set of the examples of the present invention.

[0028] Figure 6 Statistical graphs of the secretion levels of RCN1 in the cerebrospinal fluid (CSF) of the control (pressure hydrocephalus) and acute NMOSD patients in the validation set in the examples of the present invention.

[0029] Figure 7 Figure 1 is a statistical graph showing the diagnostic test results of NMOSD patients using RCN1 in an embodiment of the present invention, wherein (A) is a statistical graph related to gender, (B) is a statistical graph related to the age of the subject, (C) is a statistical graph related to the age of the patient's first attack, (D) is a statistical graph related to the patient's blood vitamin D level, (E) is a statistical graph related to blood calcium, and (F) is a statistical graph related to urine calcium.

[0030] Figure 8 Statistical graphs of the results of using RCN1 to analyze the clinical pathological characteristics of NMOSD patients in the embodiments of the present invention, (A) is a statistical graph of the correlation with EDSS score, (B) is a statistical graph of the correlation with the number of relapses, (C) is a statistical graph of the correlation with the course of disease, and (D) is a statistical graph of the correlation with AQP4 test results.

[0031] Figure 9 Graph showing the efficacy of plasma RCN1 protein level, cerebrospinal fluid RCN1 protein level, and PBMC RCN1 mRNA level in diagnosing NMOSD individually and in combination in the examples of the present invention.

[0032] Figure 10 This is a graph showing the efficacy of plasma RCN1 protein level, plasma RCN3 protein level, and plasma SDF4 protein level alone and in combination in diagnosing NMOSD in an embodiment of the present invention. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0034] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0035] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are intended to explain the present invention rather than to limit it.

[0036] Reticulin 1 (RCN1) is an endoplasmic reticulum calcium-binding protein containing six EF-hand motifs and is a member of the CREC family. RCN1 is an endoplasmic reticulum lumen protein and can be secreted outside the cell after glycosylation modification.

[0037] Example

[0038] 1. Acquisition of screening and verification samples

[0039] (1) Filter set

[0040] Ten plasma and peripheral blood mononuclear cells (PBMCs) were collected from healthy individuals undergoing physical examinations at the Department of Laboratory Medicine, Shandong University Second Hospital, as controls. Cerebrospinal fluid (CSF) samples were collected from six patients with pressure hydrocephalus at the Department of Neurology, Shandong University Second Hospital, as controls. Ten plasma and PBMC samples were collected from patients with acute NMOSD at the Department of Neurology, Qilu Hospital. Seven plasma samples were collected from patients with MS and two plasma samples were collected from patients with encephalitis at the Department of Neurology, Shandong University Second Hospital, for comparison with the aforementioned plasma and PBMC controls. Six CSF samples were collected from patients with acute NMOSD and 10 CSF samples were collected from patients with encephalitis at the Department of Neurology, Shandong University Second Hospital, for comparison with the aforementioned six CSF controls. The actual number of samples measured is shown in Table 1.

[0041] Table 1. Sample size of the screening set

[0042] Sample Type comparison NMOSD patients MS patients Encephalitis patients plasma 10 10 7 2 PBMC 10 10 0 0 CSF 6 6 0 10

[0043] (2) Validation set

[0044] Plasma and PBMC samples from 20 healthy individuals were collected from the Department of Laboratory Medicine at the Second Hospital of Shandong University as controls. Cerebrospinal fluid samples from 10 patients with hydrocephalus were collected from the Department of Neurology at the Second Hospital of Shandong University as controls. Plasma samples from 25 patients with acute NMOSD and 19 PBMC samples were collected from the Department of Neurology at the Second Hospital of Shandong University for comparison with the plasma and PBMC controls. Cerebrospinal fluid samples from 10 patients with acute NMOSD were also collected from the Department of Neurology at the Second Hospital of Shandong University for comparison with the controls. The actual number of samples measured is shown in Table 2.

[0045] Table 2 Number of samples in the validation set

[0046]

[0047]

[0048] The screening and validation sets were completely separate, and this study applied for and received a waiver of informed consent. This study was approved by the Ethics Committees of the Second Hospital of Shandong University and Qilu Hospital.

[0049] 2. Test methods

[0050] (1) The plasma detection method includes: collecting fresh peripheral blood in an anticoagulant tube, centrifuging at 2000 rpm for 10 minutes, and freezing the plasma sample at -80°C for subsequent enzyme-linked immunosorbent assay (ELISA) testing.

[0051] Then, according to the instructions of the ELISA kit, the levels of RCN1, RCN2, RCN3 and SDF4 proteins in the plasma were detected respectively.

[0052] (2) PBMC detection method includes: resuspending blood cells in 10ml of sterile PBS, gently adding to the upper layer of the same volume of lymphocyte separation solution (Ficoll solution), centrifuging at 2,000rpm for 20min to obtain peripheral blood mononuclear cells (PBMC). 7 Add 1 ml of Trizol reagent to each cell, pipette thoroughly, and freeze at -80°C for subsequent RNA extraction.

[0053] Then, RNA was extracted from PBMC and reverse transcribed into cDNA. The mRNA levels of CREC family members RCN1, RCN2, RCN3, SDF4 and CAUL (calcium cavity protein) were detected by quantitative PCR and statistical analysis was performed.

[0054] (3) The cerebrospinal fluid detection method includes: detecting the RCN1 protein content in the cerebrospinal fluid according to the ELISA kit instructions.

[0055] 3. Statistical Analysis

[0056] (1) In the screening set, plasma samples of healthy people, acute NMOSD patients, MS patients, and encephalitis patients were analyzed. Figure 1 As shown, RCN1 protein content was upregulated in the plasma of NMOSD patients with significant differences.

[0057] (2) In the screening set, the mRNA levels of CREC family members RCN1, RCN2, RCN3, SDF4, and CAUL in PBMCs of healthy subjects and patients with acute NMOSD were as follows: Figure 2 As shown in Figure 3, RCN1 mRNA was upregulated in PBMCs of NMOSD patients with significant differences.

[0058] (3) In the screening set, the cerebrospinal fluid (CSF) of patients with pressure hydrocephalus, acute NMOSD patients, and encephalitis patients, such as Figure 3 As shown, RCN1 protein content was downregulated in the CSF of NMOSD patients, which was significantly different from that of patients with pressure hydrocephalus and encephalitis.

[0059] (4) In the validation set, plasma from healthy individuals and patients with acute NMOSD, such as Figure 4 As shown in (A): RCN1 protein content is upregulated in the plasma of NMOSD patients, and there are significant differences; Figure 4 As shown in (B): RCN3 protein content is upregulated in the plasma of NMOSD patients, and there are significant differences; Figure 4 As shown in (C), SDF4 protein content was upregulated in the plasma of NMOSD patients with significant differences.

[0060] (5) In the validation set, the mRNA levels of CREC family members RCN1, RCN2, RCN3, SDF4, and CAUL in healthy subjects and patients with acute NMOSD were as follows: Figure 5 As shown: RCN1, RCN3 and SDF4 mRNA expression was upregulated in PBMCs of NMOSD patients with significant differences.

[0061] (6) In the validation set, such as Figure 6 As shown in the data, RCN1 protein content in the cerebrospinal fluid of patients with pressure hydrocephalus and acute NMOSD patients was downregulated in the CSF of NMOSD patients, and there was a significant difference.

[0062] 4. Utilizing RCN1 for diagnostic testing of NMOSD patients.

[0063] (1) In the validation set, the correlation between plasma RCN1 protein content and gender, age, age of onset, blood vitamin D, blood calcium, and urine calcium levels in patients with acute NMOSD was analyzed. Figure 7 As shown, Figure 7 (A) shows that the plasma RCN1 protein content is basically unrelated to gender; Figure 7 (B) shows that the plasma RCN1 protein content is positively correlated with the age of the subjects, with statistically significant differences; Figure 7 (C) shows that the plasma RCN1 protein content is positively correlated with the age of first onset of the patient, with statistical significance; Figure 7 (D) in the figure shows that the plasma RCN1 protein content is basically unrelated to the patient's blood vitamin D level; Figure 7 (E) shows that the plasma RCN1 protein content is basically unrelated to the patient's blood calcium; Figure 7 (F) shows that the plasma RCN1 protein content is negatively correlated with the patient's urine calcium, and there is a statistical difference.

[0064] (2) In the validation set, the correlation between plasma RCN1 protein content and EDSS score, relapse frequency, disease course and AQP4 positivity in patients with acute NMOSD was analyzed. Figure 8 As shown, Figure 8 (A) shows that the plasma RCN1 protein content is negatively correlated with the EDSS score, with statistical significance; Figure 8 (B) shows that the plasma RCN1 protein level is negatively correlated with the number of relapses, with statistical significance; Figure 8 (C) shows that the plasma RCN1 protein content is negatively correlated with the course of disease, with statistical significance; Figure 8 (D) shows that there is no significant correlation between plasma RCN1 protein content and AQP4 positivity.

[0065] 5. Diagnostic efficacy analysis.

[0066] (1) The ROC diagram of the diagnostic efficacy of plasma RCN1 protein content, cerebrospinal fluid RCN1 protein content and PBMC RCN1 mRNA level in the validation set alone and in combination for NMOSD is shown in the figure below. Figure 9 As shown, the AUC for diagnosis of plasma RCN1 protein content alone is 0.948; the AUC for diagnosis of CSF RCN1 protein content alone is 0.905; the AUC for diagnosis of RCN1 mRNA level in PBMC alone is 0.807; the AUC for diagnosis of plasma RCN1 protein content combined with CSF RCN1 protein content and PBMC RCN1 mRNA level is 0.992.

[0067] (2) The ROC diagram of the effectiveness of plasma RCN1 protein level, plasma RCN3 protein level and plasma SDF4 protein level in diagnosing NMOSD alone and in combination is shown in the following figure Figure 10 As shown, the AUC for diagnosis of plasma RCN1 protein content alone is 0.948; the AUC for diagnosis of plasma RCN3 protein content alone is 0.812; the AUC for diagnosis of plasma SDF4 protein content alone is 0.880; and the AUC for combined diagnosis of plasma RCN1, plasma RCN3 and plasma SDF4 protein content is 0.998.

[0068] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. Use of a reagent for detecting RCN1 and its related biological materials in the preparation of products for screening, diagnosing, detecting, monitoring NMOSD or predicting the progression of NMOSD.

2. The use according to claim 1, characterized in that The RCN1 is derived from plasma or cerebrospinal fluid.

3. The use according to claim 1, characterized in that The relevant biological materials include mRNA of RCN1.

4. The use according to claim 3, characterized in that The RCN1 mRNA is derived from peripheral blood mononuclear cells.

5. The use according to claim 1, characterized in that The product includes reagents for detecting RCN3 and SDF4.

6. The use according to claim 5, characterized in that The RCN3 and SDF4 are derived from plasma.

7. A product for screening, diagnosing, detecting, monitoring NMOSD or predicting the progression of NMOSD, characterized in that: It comprises substances for detecting RCN1 and related biological materials in a sample to be tested based on a high-throughput sequencing method and / or a quantitative PCR method and / or a probe hybridization method.

8. The product according to claim 7, characterized in that The product includes reagents for detecting RCN3 and SDF4.

9. A system for screening, diagnosing, detecting, monitoring, or predicting the progression of NMOSD, characterized in that: include: An analysis module, for determining the expression level of a detection substance selected from RCN1 and its related biological materials in a test sample of a subject; The evaluation module is used to judge the condition of the subject according to the expression level of RCN1 and its related biological materials determined by the analysis module.

10. The system according to claim 9, characterized in that It includes a combined detection module, which is used to detect the detection substances of the expression levels of RCN3 and SDF4 and related biological materials in the test samples of the subjects.

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