Application of human protocadherin 18 as a biomarker in the preparation of products for diagnosis, auxiliary diagnosis or progression prediction of antiphospholipid syndrome

Abstract

This invention provides the application of human protocadherin 18 (PCDH18) as a biomarker in the preparation of products for the diagnosis, auxiliary diagnosis, or progression prediction of antiphospholipid syndrome (APS), belonging to the field of in vitro diagnostic technology. In this invention, detecting serum PCDH18 levels in patients is helpful in diagnosing APS and is specific to this disease, and can be used to differentiate APS from other connective tissue diseases. Furthermore, APS patients have higher serum PCDH18 levels compared to asymptomatic aPL-positive patients. Therefore, PCDH18 testing in aPL-positive individuals is helpful for the early diagnosis and intervention of APS. Additionally, serum PCDH18 levels in OAPS patients are significantly higher than in TAPS patients, suggesting that PCDH18 can serve as a biomarker for differentiating or assisting in the differentiation of TAPS and OAPS.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic technology, and in particular relates to the application of human procadherin 18 as a biomarker in the preparation of products for the diagnosis, auxiliary diagnosis or progression prediction of antiphospholipid syndrome. Background Technology

[0002] Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterized by recurrent arterial or venous thrombosis or habitual abortion, accompanied by persistent positive antiphospholipid antibodies (aPLs). When thrombosis is the primary clinical manifestation, it is called thrombotic APS (TAPS), and when pathological pregnancy is the primary clinical feature, it is called obstetric APS (OAPS). Currently, there are no biomarkers in clinical practice to differentiate between TAPS and OAPS. Summary of the Invention

[0003] The purpose of this invention is to provide the application of human cadherin 18 as a biomarker in the preparation of products for the diagnosis, auxiliary diagnosis or progression prediction of antiphospholipid syndrome. Human cadherin 18 can be used as a biomarker for clinical differentiation between TAPS and OAPS.

[0004] This invention provides the application of human procadherin 18 as a biomarker in the preparation of products for the diagnosis, auxiliary diagnosis or progression prediction of antiphospholipid syndrome.

[0005] Preferably, the application includes the quantitative detection of human procadherin 18 in biological samples obtained from individuals, thereby diagnosing, assisting in diagnosis, or predicting the progression of phospholipid syndrome.

[0006] Preferably, the biological sample includes serum.

[0007] This invention also provides the application of human procadherin 18 as a biomarker in the preparation of products for the identification or auxiliary identification of thrombotic APS and obstetric APS.

[0008] This invention also provides the application of human procadherin 18 as a biomarker in the preparation of products for the identification or auxiliary identification of antiphospholipid syndrome and asymptomatic antiphospholipid antibody positivity.

[0009] This invention also provides the use of human procadherin 18 as a biomarker in the preparation of products for the identification or auxiliary identification of antiphospholipid syndrome and other connective tissue diseases.

[0010] Preferably, the other connective tissue diseases include rheumatoid arthritis and / or systemic lupus erythematosus.

[0011] Preferably, the amino acid sequence of the human procadherin 18 is shown in SEQ ID NO.1.

[0012] Preferably, the product includes reagents or kits.

[0013] Preferably, the kit includes an ELISA detection kit.

[0014] This invention provides the application of human protocadherin 18 (PCDH18) as a biomarker in the preparation of products for the diagnosis, auxiliary diagnosis, or progression prediction of antiphospholipid syndrome (APS). This invention uses PCDH18 as a biomarker for the diagnosis, auxiliary diagnosis, or progression prediction of APS. Serum PCDH18 levels in APS patients are significantly higher than in disease controls and healthy controls, and ROC curves indicate good diagnostic performance. Therefore, detecting serum PCDH18 levels in patients is helpful in diagnosing APS and is specific to this disease, and can be used to differentiate APS from other connective tissue diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Furthermore, compared with asymptomatic aPL-positive patients, APS patients have higher serum PCDH18 levels, and after adjusting for factors such as aCL positivity, PCDH18 can still predict the risk of progression to APS in asymptomatic aPL-positive patients. Therefore, PCDH18 testing in aPL-positive individuals is helpful for the early diagnosis and intervention of APS. In addition, serum PCDH18 levels were significantly higher in OAPS patients than in TAPS patients, suggesting that PCDH18 can serve as a marker for differentiating or assisting in the differentiation between thrombotic APS (TAPS) and obstetric APS (OAPS). Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The distribution of serum PCDH18 in APS patients, asymptomatic aPL-positive patients, disease controls, and healthy controls is shown in the figure; DC (disease control): disease control; HC (healthy control): healthy control.

[0017] Figure 2The distribution of serum PCDH18 in patients with thrombotic APS and obstetric APS is shown in the figure. Among them, OAPS (obstetric APS): obstetric antiphospholipid syndrome; TAPS (thrombotic APS): thrombotic antiphospholipid syndrome.

[0018] Figure 3 ROC curves for serum PCDH18 (TAPS vs. HC, OAPS vs. HC);

[0019] Figure 4 ROC curves for serum PCDH18 (APS vs. HC);

[0020] Figure 5 To validate the ROC curve of serum PCDH18 (APS vs. HC). Detailed Implementation

[0021] This invention provides the application of human protocadherin 18 (PCDH18) as a biomarker in the preparation of products for the diagnosis, auxiliary diagnosis or progression prediction of antiphospholipid syndrome.

[0022] In this invention, serum PCDH18 levels in APS patients were significantly higher than those in disease controls and healthy controls, and the ROC curve indicated good diagnostic performance. Therefore, detecting serum PCDH18 levels in patients is helpful in diagnosing APS and is specific to this disease, and can be used to differentiate APS from other connective tissue diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Furthermore, compared with asymptomatic aPL-positive patients, APS patients had higher serum PCDH18 levels, and after adjusting for factors such as aCL positivity, PCDH18 could still predict the risk of asymptomatic aPL-positive patients progressing to APS. Therefore, PCDH18 testing in aPL-positive individuals is helpful for the early diagnosis and intervention of APS. Additionally, serum PCDH18 levels in OAPS patients were significantly higher than those in TAPS patients, suggesting that PCDH18 can serve as a marker for differentiating or assisting in the differentiation of TAPS and OAPS.

[0023] In this invention, serum PCDH18 ≥ 12.5 pg / mL is defined as PCDH18 positive. After adjusting for thrombocytopenia, aCL(+), and aβ2GPI(+) using multivariate regression analysis, it was found that PCDH18(+) can still predict the progression of aPL positive patients to APS (p = 0.009).

[0024] In the specific implementation of this invention, the application includes quantitative detection of PCDH18 in biological samples obtained from individuals, thereby diagnosing, assisting in diagnosis, or predicting the prognosis of the progression of antiphospholipid syndrome.

[0025] In the specific implementation of this invention, the biological sample includes serum.

[0026] In one embodiment of the present invention, the quantitative detection of human protocadherin 18 in serum obtained from an individual includes: detecting the level of PCDH18 in biological samples obtained from the individual using an ELISA kit; the ELISA kit is purchased from Shanghai Jiwei and has the specification GV-E25378.

[0027] This invention also provides the application of human procadherin 18 as a biomarker in the preparation of products for the identification or auxiliary identification of thrombotic APS and obstetric APS.

[0028] In this invention, the level of PCDH18 in serum samples from obstetric APS patients was significantly higher than that in thrombotic APS patients. The AUC of PCDH18 in OAPS was 0.829 (95% CI: 0.742–0.916, p<0.0001), and in TAPS it was 0.801 (95% CI: 0.700–0.902, p<0.0001).

[0029] This invention also provides the application of human procadherin 18 as a biomarker in the preparation of products for the identification or auxiliary identification of antiphospholipid syndrome and asymptomatic antiphospholipid antibody positivity.

[0030] In this invention, APS patients have higher serum PCDH18 levels compared to asymptomatic aPL-positive patients.

[0031] This invention also provides the use of human procadherin 18 as a biomarker in the preparation of products for the identification or auxiliary identification of antiphospholipid syndrome and other connective tissue diseases.

[0032] In the specific implementation of this invention, the other connective tissue diseases include rheumatoid arthritis and / or systemic lupus erythematosus. Serum PCDH18 levels in APS patients are significantly higher than in patients with other connective tissue diseases. In the specific implementation of this invention, the optimal cutoff value for distinguishing between APS patients and healthy individuals is 12.50 pg / mL, with a sensitivity of 97.2%, specificity of 65.9%, area under the curve (AUC) of 0.834 (95% CI: 0.749–0.919, p<0.0001), and Youden's index of 0.659. The optimal cutoff value for distinguishing between APS and other connective tissue diseases is 6.89 pg / mL, with a sensitivity of 48.7%, specificity of 98.6%, AUC of 0.708 (95% CI: 0.586–0.831, p=0.0003), and Youden's index of 0.399. It is evident that PCDH18 levels have high value in the diagnosis of APS.

[0033] In the specific implementation of this invention, the amino acid sequence of human procadherin 18 is as shown in SEQ ID NO.1, specifically:

[0034]

[0035] In the specific implementation of this invention, the product includes reagents or kits.

[0036] In the specific implementation of this invention, the kit includes an ELISA detection kit.

[0037] To further illustrate the present invention, the application of human protocadherin 18 as a biomarker provided by the present invention in the preparation of products for the diagnosis, auxiliary diagnosis or progression prediction of antiphospholipid syndrome is described in detail below with reference to the accompanying drawings and embodiments. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0038] Example 1

[0039] We collected data from 71 patients with acute thrombotic syndrome (APS) who visited Peking University People's Hospital between January 2019 and June 2022. The mean age was 39.28 years. Among them, there were 21 cases of thrombotic APS (TAPS) and 50 cases of obstetric APS (OAPS). Among the 71 APS patients, 13 had thrombocytopenia, 14 were positive for anticardiolipin antibody (aCL), 54 were positive for anti-β2-glycoprotein I antibody (aβ2GPI), and 20 were positive for lupus anticoagulant (LA).

[0040] Based on the age and sex of APS patients, 39 disease controls (DCs) were matched, including 7 patients with rheumatoid arthritis and 32 patients with systemic lupus erythematosus. In addition, 51 asymptomatic aPL-positive patients and 48 healthy controls (HCs) were included. None of the aPL-positive patients, DCs, or HCs had thrombosis or morbid pregnancy. Basic information on APS patients and controls is detailed in Table 1.

[0041] Table 1. Basic information of APS patients, aPL-positive patients, disease controls, and healthy controls.

[0042]

[0043] This study was approved by the Ethics Committee of Peking University People's Hospital (Approval No.: 2020PHB247), and the patients signed informed consent forms. All enrolled APS patients met the 2006 Sydney criteria. General information (age, sex), clinical information (thrombosis, morbid pregnancy, thrombocytopenia, smoking, hypertension, diabetes, coronary heart disease, etc.), and laboratory tests (complete blood count, blood biochemistry, coagulation indicators, inflammatory indicators, immunological indicators, etc.) were collected from the patients. Thrombotic events were confirmed by vascular ultrasound, angiography, or pathology. Morbid pregnancy met any of the following criteria: (1) ≥1 morphologically normal stillbirth at ≥10 weeks of gestation that could not be explained by other causes; (2) ≥1 morphologically normal preterm birth at <34 weeks of gestation due to epilepsy, preeclampsia, or severe placental insufficiency; (3) ≥3 spontaneous abortions at <10 weeks of gestation that could not be explained by other causes.

[0044] The concentration of PCDH18 in serum samples was detected using the Human Protocadherin 18 (PCDH18) ELISA Kit (Shanghai Jiwei, GV-E25378).

[0045] The operation steps are as follows: (1) Equilibrate at room temperature for 20 min; (2) Add 50 μL of different concentrations of standard to each standard well, do not add to the blank well, and add 50 μL of the sample to be tested to the sample well; (3) Except for the blank well, add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to each well, seal the reaction well with sealing film, and incubate at 37℃ for 60 min; (4) Discard the liquid, pat dry on absorbent paper, fill each well with washing buffer (350 μL), let stand for 1 min, shake off the washing buffer, pat dry on absorbent paper, and repeat the washing process 5 times; (5) Add 50 μL of substrate A and B to each well, and incubate at 37℃ in the dark for 15 min; (6) Add 50 μL of stop solution to each well, and within 15 min, measure the OD value of each well at a wavelength of 450 nm.

[0046] Statistical analysis and graphing were performed using GraphPadPrism 10.1.2. Categorical variables underwent chi-square tests, and continuous variables were tested for normality. If a variable followed a normal distribution, it was considered... This indicates that comparisons between two groups are performed using an independent samples t-test (for homogeneous variances) or Welch's t-test (for unequal variances), while comparisons among multiple groups are performed using an ANOVA test; if the distribution does not follow a normal distribution, the following methods are used: The Mann-Whitney U test was used for comparisons between two groups, and the Kruskal-Wallis H test was used for comparisons among multiple groups. If the data followed a normal distribution, Spearman correlation analysis was used; otherwise, Pearson correlation analysis was used. The sensitivity and specificity of the diagnostic indicators were evaluated using receiver operating characteristic (ROC) curves. A p-value < 0.05 was considered statistically significant.

[0047] The serum PCDH18 concentrations of APS patients, asymptomatic aPL-positive patients, disease controls (DC), and healthy controls (HC) were compared. The results are as follows: The serum PCDH18 concentrations of APS patients were significantly higher than those of DC [55.69 (30.41, 75.42) pg / mL vs 37.31 (5.00, 72.20) pg / mL, U = 784, p < 0.001] and HC [55.69 (30.41, 75.42) pg / mL vs 22.78 (5.00, 31.50) pg / mL, U = 566, p < 0.001]. There was no statistically significant difference between DC and HC [37.31 (5.00, 72.20) pg / mL vs 22.78 (5.00, 31.50) pg / mL, U = 766, p = 0.119]. Serum PCDH18 concentrations in APS patients were significantly higher than those in aPL-positive patients [55.69 (30.41, 75.42) pg / mL vs 43.32 (5.14, 65.77) pg / mL, U = 1399, p = 0.032]. PCDH18 levels in aPL-positive patients were significantly higher than those in DC [43.32 (15.14, 65.77) pg / mL vs 37.31 (5.00, 72.20) pg / mL, U = 748.5, p = 0.043] and HC [43.32 (15.14, 65.77) pg / mL vs 22.78 (5.00, 31.50) pg / mL, U = 661.5, p < 0.001]. In the example of 55.69 (30.41, 75.42), 55.69 is the mean, 30.41 is the first quartile (25th percentile), and 75.42 is the third quartile (75th percentile). U is the statistical value; the smaller the U value, the greater the difference between the two groups. In the Mann-Whitney U test, the p-value can be calculated from the U value, and the p-value is used to measure whether the observed difference is statistically significant. See [link to relevant documentation]. Figure 1 .

[0048] Comparing serum PCDH18 concentrations in patients with thrombotic APS (TAPS) and obstetric APS (OAPS), it was found that serum PCDH18 levels in obstetric APS patients were significantly higher than those in thrombotic APS patients (43.03±27.88 pg / mL vs 61.01±34.58 pg / mL, p=0.039). (See attached image) Figure 2 .

[0049] The diagnostic performance of PCDH18 was evaluated using receiver operating characteristic (ROC) curves. See results below. Figure 2 The results showed that the optimal cutoff value for distinguishing between APS and HC was 12.50 pg / mL, with a sensitivity of 97.2%, a specificity of 65.9%, an area under the curve (AUC) of 0.834 (95% CI: 0.749–0.919, p < 0.0001), and a Youden index of 0.659. (See [link to relevant documentation]). Figure 4 The optimal cutoff value for differentiating between APS and DC was 6.89 pg / mL, with a sensitivity of 48.7%, specificity of 98.6%, AUC of 0.708 (95% CI: 0.586–0.831, p = 0.0003), and Youden's index of 0.399. This indicates that PCDH18 levels have high value in the diagnosis of APS. Comparing the AUCs of TAPS and OAPS, the AUC of PCDH18 in OAPS was 0.829 (95% CI: 0.742–0.916, p < 0.0001), while in TAPS it was 0.801 (95% CI: 0.700–0.902, p < 0.0001). (See...) Figure 3 Serum PCDH18 ≥ 12.5 pg / mL was defined as PCDH18 positive. After adjusting for thrombocytopenia, aCL(+), and aβ2GPI(+) using multivariate regression analysis, it was found that PCDH18(+) could still predict the progression of aPL positive patients to APS (p = 0.009), as shown in Table 2.

[0050] Table 2. Logistic regression analysis results of asymptomatic aPL-positive patients developing APS.

[0051]

[0052] Verification Example 1

[0053] Another 40 APS patients and 20 healthy controls were selected as the validation set (basic information is shown in Table 3). The concentration of PCDH18 in serum samples was measured, and ROC curves were plotted. Figure 5The experimental methods and statistical analysis were as described in the above examples. The results showed an AUC of 0.0624 (95% CI: 0.6953–0.9397), a cutoff value of 13.1 pg / mL, a sensitivity of 87.5%, a specificity of 70%, and a Youden index of 0.575. Therefore, in the validation sample set, PCDH18 also demonstrated good ability to distinguish between APS and healthy individuals.

[0054] Table 3. Basic information of APS patients and healthy controls in the validation set.

[0055]

[0056] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of human procadherin 18 as a biomarker in the preparation of products for the diagnosis or auxiliary diagnosis of antiphospholipid syndrome.

2. The application according to claim 1, characterized in that, The applications include the quantitative detection of human procadherin 18 in biological samples obtained from individuals, thereby diagnosing or assisting in the diagnosis of antiphospholipid syndrome.

3. The application according to claim 2, characterized in that, The biological samples include serum.

4. The application according to any one of claims 1 to 3, characterized in that, The amino acid sequence of the human protocadherin 18 is shown in SEQ ID NO.

1.

5. The application according to any one of claims 1 to 3, characterized in that, The products include reagents or kits.

6. The application according to claim 5, characterized in that, The kit includes an ELISA detection kit.

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