Diagnostic markers for systemic lupus erythematosus and their applications

Abstract

The application provides a diagnostic marker for systemic lupus erythematosus and application thereof, and belongs to the technical field of biological medicine. The diagnostic marker is at least one protein in PDGFA, APOD, CFHR5, AMDHD1 and SCGB3A1; the amino acid sequence of PDGFA is shown as SED ID NO.1; the amino acid sequence of APOD is shown as SED ID NO.2; the amino acid sequence of CFHR5 is shown as SED ID NO.3; the amino acid sequence of AMDHD1 is shown as SED ID NO.4; and the amino acid sequence of SCGB3A1 is shown as SED ID NO.5. The application proposes that the above-mentioned five proteins are used as biomarkers for SLE disease diagnosis, and have the advantages of high accuracy, high sensitivity and high specificity, and provide a new target for the diagnosis and intervention improvement of SLE patients.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to diagnostic markers for systemic lupus erythematosus and their applications. Background Technology

[0002] Systemic lupus erythematosus (SLE) is an autoimmune disease affecting multiple systems and organs, characterized by the presence of various autoantibodies. Its pathogenesis is complex, and its clinical manifestations exhibit high heterogeneity. Currently, SLE diagnosis relies on a variety of clinical and laboratory biomarkers, some of which have high specificity, such as anti-dsDNA antibodies, anti-Sm antibodies, and anti-nucleosome antibodies (AnuA). However, some patients test negative or show fluctuating titers, resulting in low sensitivity. Conventional diagnostic biomarkers currently lack both high sensitivity and specificity, thus failing to meet the needs of precision medicine. Summary of the Invention

[0003] Therefore, the present invention aims to provide diagnostic biomarkers for systemic lupus erythematosus and their applications, in order to solve at least one of the technical problems in the background art.

[0004] This invention is implemented as follows:

[0005] The first aspect of the present invention provides a diagnostic biomarker for systemic lupus erythematosus, wherein the diagnostic biomarker is at least one protein selected from PDGFA, APOD, CFHR5, AMDHD1, and SCGB3A1;

[0006] The PDGFA is platelet-derived growth factor A, and its amino acid sequence is shown in SED ID NO.1;

[0007] The APOD is apolipoprotein D, and its amino acid sequence is shown in SED ID NO.2;

[0008] The CFHR5 is complement factor H-related protein 5, and its amino acid sequence is shown in SED ID NO.3;

[0009] The AMDHD1 contains an amide hydrolase domain 1, and its amino acid sequence is shown in SED ID NO.4;

[0010] The SCGB3A1 is a member of the secretory globulin family 3A, and its amino acid sequence is shown in SED ID NO.5.

[0011] Preferably, the diagnostic biomarker is a combination of at least two proteins selected from PDGFA, APOD, CFHR5, AMDHD1, and SCGB3A1.

[0012] Preferably, the diagnostic biomarker is a combination of at least three proteins selected from PDGFA, APOD, CFHR5, AMDHD1, and SCGB3A1.

[0013] Preferably, the diagnostic biomarker is a combination of at least four proteins selected from PDGFA, APOD, CFHR5, AMDHD1, and SCGB3A1.

[0014] Preferably, the diagnostic biomarkers are all combinations of PDGFA, APOD, CFHR5, AMDHD1, and SCGB3A1.

[0015] A second aspect of the present invention provides the use of a reagent for detecting the expression level of the above-mentioned diagnostic markers for systemic lupus erythematosus in the preparation of products for diagnosing systemic lupus erythematosus.

[0016] Preferably, the product is based on mass spectrometry to detect the expression level of the diagnostic marker in the blood.

[0017] Preferably, the product is a kit or reagent.

[0018] A third aspect of the present invention provides a product for diagnosing systemic lupus erythematosus, the product comprising a reagent for detecting the expression levels of the diagnostic markers for systemic lupus erythematosus described above.

[0019] Preferably, the product is based on mass spectrometry to detect the expression level of the diagnostic marker in blood; the product is a kit or reagent.

[0020] This invention identifies significant differences in the expression levels of five proteins—PDGFA, APOD, CFHR5, AMDHD1, and SCGB3A1—in blood samples from SLE patients and healthy individuals. Therefore, it proposes these five proteins, individually or in combination, as biomarkers for SLE diagnosis, offering advantages of high accuracy, high sensitivity, and high specificity, providing new targets for SLE diagnosis and intervention. Developing corresponding auxiliary early diagnostic reagents and kits based on these five protein biomarkers, individually or in combination, has broad research value and clinical applications, greatly facilitating early screening, clinical diagnosis, and intervention. Attached Figure Description

[0021] Figure 1 Box plots showing the differential expression of five proteins in the blood of the SLE group and the HC group in the experimental set;

[0022] Figure 2 ROC curves of five proteins used individually as biomarkers in the experimental group in the SLE and HC groups;

[0023] Figure 3ROC curves for the SLE group and HC group, showing any two combinations of the five proteins in the experimental set as biomarkers.

[0024] Figure 4 ROC curves for the SLE group and HC group, showing any combination of three of the five proteins in the experimental set as biomarkers.

[0025] Figure 5 ROC curves for the SLE group and HC group, showing any combination of four of the five proteins in the experimental set as biomarkers.

[0026] Figure 6 ROC curves of all five protein combinations used as biomarkers in the experimental group were obtained in the SLE and HC groups.

[0027] Figure 7 To validate the box plots showing the differential expression of five proteins in the blood of the SLE group and the HC group;

[0028] Figure 8 To verify the ROC curves of the five proteins as biomarkers in the SLE and HC groups;

[0029] Figure 9 To verify the ROC curves of any two combinations of the five proteins in the sample as biomarkers in the SLE and HC groups;

[0030] Figure 10 To verify the ROC curves of any three combinations of the five proteins in the sample as biomarkers in the SLE and HC groups;

[0031] Figure 11 To verify the ROC curves of any four combinations of the five proteins in the sample as biomarkers in the SLE and HC groups;

[0032] Figure 12 To verify the ROC curves of all five protein combinations as biomarkers in the SLE and HC groups. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0034] I. Experimental Subjects

[0035] The experimental set included 138 patients with systemic lupus erythematosus (SLE) as the SLE group and 58 healthy individuals without SLE as the HC group; the validation set included 51 patients with SLE as the SLE group and 42 healthy individuals without SLE as the HC group; the characteristic information is shown in Table 1 below.

[0036] Table 1

[0037]

[0038] Some patients with systemic lupus erythematosus lack clinical information on indicators such as occult blood in urine, protein in urine, anti-double-stranded DNA antibody, antinuclear antibody, and anti-Sm antibody. Therefore, when calculating the positive rate of the above indicators, the total number of patients with clinical information on the above indicators is used as the benchmark.

[0039] II. Screening for Differentially Expressed Proteins

[0040] 1. Enzymatic hydrolysis

[0041] (1) Place the blood sample in a refrigerated high-speed centrifuge, centrifuge at 12000g for 10 min at 4°C to remove cell debris, and transfer the supernatant to a new centrifuge tube;

[0042] (2) Take 50µL of blood sample after high-speed centrifugation into the pre-washed magnetic nanomaterial (PTM-00F13303, from Hangzhou Jingjie Biotechnology Co., Ltd., PTM Bio), and incubate at 1200rpm and 37℃ for 1 hour on a constant temperature mixer.

[0043] (3) After incubation, centrifuge at room temperature at 10000g for 5 minutes, discard the supernatant and collect the magnetic beads;

[0044] (4) Add 300µL of washing buffer to the centrifuged magnetic bead precipitate and wash the magnetic beads by horizontal shaking at 1200g for 5min.

[0045] (5) Repeat step (4) twice;

[0046] (6) Add 70 μL of enzyme digestion buffer to the washed magnetic beads, mix well, and heat in a metal bath at 95°C for 10 min;

[0047] (7) After heating, wait for the sample to return to room temperature, add trypsin at a final concentration of 20 ng / μL, and incubate overnight at 37°C.

[0048] (8) After the enzymatic hydrolysis is completed the next day, dithiothreitol (DTT) is added to make the final concentration 5mM, and the mixture is reduced at 56℃ for 30min.

[0049] (9) Add iodoacetamide (IAM) to make a final concentration of 11 mM, and incubate at room temperature in the dark for 15 min.

[0050] 2. Desalination

[0051] (1) Acidify the enzymatically hydrolyzed peptides with 10% trifluoroacetic acid (TFA) to pH=2~3, centrifuge at 12000g for 10min at room temperature, and transfer the supernatant to a new ep tube;

[0052] (2) Activation: Add 50 μL of activation solution to StageTip and centrifuge at 1500g for 1 min;

[0053] (3) Equilibration: Add 50 μL of desalting solution to StageTip and centrifuge at 1500g for 1 min;

[0054] (4) Repeat step (3) once;

[0055] (5) Sample loading: Discard the waste liquid in the ep tube, add the acidified peptide to the StageTip, and centrifuge at 1500g for 1min;

[0056] (6) Desalination: Add 50 μL of desalination solution to StageTip and centrifuge at 1500g for 1 min;

[0057] (7) Repeat step (6) once.

[0058] (8) Elution: Put the StageTip into a new ep tube, add 20 μL of elution buffer into the StageTip, and centrifuge at 750g for 1 min;

[0059] (9) Repeat step (8) once, combine the two eluents, freeze dry and set aside.

[0060] 3. Liquid chromatography-mass spectrometry analysis

[0061] Peptides were dissolved in the mobile phase for liquid chromatography and then separated using a Vanquish Neo ultra-high performance liquid chromatography system. Mobile phase A was an aqueous solution containing 0.1% formic acid; mobile phase B was an aqueous solution containing 0.1% formic acid and 80% acetonitrile. The liquid phase gradient settings were as follows: 0 min–1.6 min, 4% B–22.5% B; 1.6 min–2.0 min, 22.5% B–35% B; 2.0 min–2.6 min, 35% B–55% B; 2.6 min–2.7 min, 55% B–99% B; 2.7 min–6.8 min, 99% B; 6.8 min–7.6 min, 99% B, with the flow rate maintained at 300 nmol / min.

[0062] Peptides were separated by an ultra-high performance liquid chromatography (UHPLC) system and then injected into an NSI ion source for ionization before being analyzed by an Orbitrap Astral mass spectrometer. The ion source voltage was set to 1900V. The precursor peptide ion was detected and analyzed using an Orbitrap detector, while secondary fragment ions were detected and analyzed using an Astral detector. The primary mass spectrometry scan range was set to 380 m / z to 980 m / z, with a scan resolution of 240,000 m / z. The secondary mass spectrometry scan range had a fixed starting point of 150 m / z, with a secondary scan resolution of 80,000 m / z. Data acquisition was performed using a data-independent scan (DIA) procedure, where multiple consecutive windows of peptide ions were introduced into the HCD collision cell after the primary scan, fragmented using 25% of the fragmentation energy, and then analyzed sequentially by secondary mass spectrometry. To improve the efficiency of the mass spectrometer, automatic gain control (AGC) was set to 500%, and the maximum injection time was set to 3 ms.

[0063] 4. Database search

[0064] DIA data was retrieved using the DIA-NN (v 1.8) search engine with default parameters. The database was Homo_sapiens_9606_SP_20230103.fasta (20389 sequences), with Trypsin / P restriction enzyme digestion set to 1 and the maximum missed digestion count set to 1. Fixed modifications were N-term M excision and C carbamidomethylation. A theoretical spectral library was constructed using deep learning algorithms, and a reverse library was added to calculate the false positive rate (FDR) caused by random matching; the FDR for predictor identification was set to 1%.

[0065] III. Verifying Diagnostic Efficiency

[0066] Figure 1 and Figure 7 The figures show a comparison of the expression levels of five proteins in the blood of SLE and HC subjects in the experimental and validation sets, respectively. The relationship between the differences in the expression levels of these five proteins in the blood of SLE and HC subjects and clinical parameters was evaluated using statistical tests and regression analysis. Diagnostic efficacy was evaluated using the receiver operating characteristic (ROC) curve. A diagnostic indicator was considered "highly accurate" when the area under the ROC curve (AUC) was ≥0.9, "accurate" when 0.8 ≤ AUC < 0.9, and "moderately accurate" when 0.7 ≤ AUC < 0.8.

[0067] The five proteins are PDGFA, APOD, CFHR5, AMDHD1, and SCGB3A1. PDGFA is platelet-derived growth factor A, and its amino acid sequence is shown in SED ID NO.1. APOD is apolipoprotein D, and its amino acid sequence is shown in SED ID NO.2. CFHR5 is complement factor H-related protein 5, and its amino acid sequence is shown in SED ID NO.3. AMDHD1 is an amino acid-containing amide hydrolase domain 1, and its amino acid sequence is shown in SED ID NO.4. SCGB3A1 is a member of the secretory globulin family 3A, and its amino acid sequence is shown in SED ID NO.5.

[0068] The above five proteins, alone or in combination, were used as serum diagnostic markers to distinguish between the SLE and HC groups. The ROC curve results for SLE and HC in the experimental set are shown below. Figures 2 to 6 Verify the ROC curve results of SLE and HC in the set. Figures 8 to 12 As shown, the AUC, sensitivity, and specificity of the experimental and validation sets are shown in Tables 2 to 6.

[0069] Table 2

[0070]

[0071] Table 3

[0072]

[0073] Table 4

[0074]

[0075] Table 5

[0076]

[0077] Table 6

[0078]

[0079] Depend on Figure 1 and Figure 7 As can be seen, the expression levels of APOD and SCGB3A1 in the blood of patients with systemic lupus erythematosus (SLE) were higher than those in healthy individuals (HC), and the differences were highly significant (p < 0.001); the expression levels of PDGFA, CFHR5, and AMDHD1 in the blood of SLE patients were lower than those in healthy individuals (HC), and the differences were highly significant (p < 0.001).

[0080] Figure 2 , Figure 8The results in Table 2 show that the differentially expressed proteins can effectively distinguish between SLE and HC on their own (AUC>0.8), but their sensitivity and specificity need to be improved. The sensitivity or specificity of some proteins in diagnosis is <70%.

[0081] like Figures 3 to 6 , Figures 9 to 12 As shown in Tables 3 to 6, any combination of two, three, four, or all of the five proteins can effectively distinguish between SLE and HC as biomarkers, and their AUC, sensitivity, or specificity are significantly better than those of individual proteins. The advantage of combinations of three or more proteins is even more obvious, with a significant improvement in AUC, sensitivity, or specificity.

[0082] Experimental results show that the five proteins can serve as blood diagnostic biomarkers to distinguish healthy individuals from those with SLE, and can be used to develop diagnostic products for systemic lupus erythematosus, such as kits and reagents. This combination of biomarkers exhibits higher sensitivity, specificity, and accuracy. It provides important evidence for further clinical research and offers new insights into SLE diagnosis and treatment.

[0083] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. Use of a diagnostic marker for systemic lupus erythematosus in the manufacture of a product for diagnosing systemic lupus erythematosus, characterized in that, The diagnostic marker is at least one protein in AMDHD1, SCGB3A1; The AMDHD1 is amide hydrolase domain 1, and its amino acid sequence is shown as SED ID NO. 4; The SCGB3A1 is secretoglobin family 3A member 1, and its amino acid sequence is shown as SED ID NO.

5.

2. Use according to claim 1, characterized in that, The diagnostic marker further comprises at least one protein in PDGFA, APOD, CFHR5; The PDGFA is platelet-derived growth factor A, and its amino acid sequence is shown as SED ID NO. 1; The APOD is apolipoprotein D, and its amino acid sequence is shown as SED ID NO. 2; The CFHR5 is complement factor H-related protein 5, and its amino acid sequence is shown as SED ID NO.

3.

3. Use according to claim 2, characterized in that, The diagnostic marker is a combination of at least three proteins in PDGFA, APOD, CFHR5, AMDHD1, and SCGB3A1.

4. Use according to claim 3, characterized in that, The diagnostic marker is a combination of at least four proteins in PDGFA, APOD, CFHR5, AMDHD1, and SCGB3A1.

5. Use according to claim 4, characterized in that, The diagnostic marker is a combination of all of PDGFA, APOD, CFHR5, AMDHD1, and SCGB3A1.

6. Use according to claim 1, characterized in that, The product detects the expression amount of the diagnostic marker in blood based on mass spectrometry.

7. Use according to claim 6, characterized in that, The product is a kit or a reagent.

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