Molecular diagnostic biomarkers for amyotrophic lateral sclerosis (ALS) and their applications

Abstract

This invention discloses a molecular diagnostic biomarker for amyotrophic lateral sclerosis (ALS) and its application. The ALS molecular diagnostic biomarker includes Ng and / or GP1bβ. This invention demonstrates that Ng and / or GP1bβ proteins are specifically and significantly upregulated in plasma. The area under the curve (AUC) for Ng alone in diagnosing ALS reaches 80.6%, the AUC for GP1bβ alone reaches 71%, and the AUC for the combined diagnosis of ALS by Ng and GP1bβ reaches 94%, with a specificity of 85%. Furthermore, Ng expression is significantly correlated with the progression of ALS, with a PCC of 0.57 (P-value = 3.4 × 10⁻⁶). ‑6 In particular, Ng expression was significantly correlated with the early progression rate of ALS (PCC = 0.45 (8.8 × 10⁻⁶)). ‑3 This indicates that Ng can also serve as a molecular diagnostic marker for the rate of ALS progression, used to predict patient survival.

Description

Technical Field

[0001] This invention relates to the field of disease diagnosis and prevention technology, and more specifically, to molecular diagnostic markers for amyotrophic lateral sclerosis (ALS) and their applications. Background Technology

[0002] Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease characterized by the progressive loss of motor neurons (MNs) in the cortex, medulla oblongata, and spinal cord. ALS typically leads to death within 3 to 5 years of onset. Approximately 450,000 people worldwide suffer from ALS, with about 10% having a family history of the disease and the remainder being sporadic. However, the underlying mechanisms of ALS remain unclear, and there are no diagnostic tests or effective treatments. Strong biomarkers will help improve diagnostic and prognostic approaches and provide insights into the pathogenesis of the disease.

[0003] There are still few reported diagnostic biomarkers for amyotrophic lateral sclerosis (ALS). For example, a Chinese patent discloses a biomarker for the early diagnosis of sporadic ALS and Parkinson's disease and its application, wherein the biomarker is lncRNAs. Iddo Magen et al. (2021) disclosed that miR-181 is a prognostic biomarker for amyotrophic lateral sclerosis (Hornstein E, Magen I, Coenen-Stass A, et al. CircμLating miR-181 is aprognostic biomarker for amyotrophic lateral sclerosis[J]. Nature Neuroscience.); Léon Beyer et al. (2020) disclosed the application of TDP-43 protein as a structural biomarker in the diagnosis of amyotrophic lateral sclerosis (Léon Beyer, René Günther, Koch JC, et al. TDP as structure-based biomarker in amyotrophic lateral sclerosis[J]. Annals of Clinical and Translational Neurology.). Therefore, there is a need to provide more reliable ALS biomarkers for clinical selection.

[0004] Neurogranin (Ng) is a newly discovered brain-specific protein composed of 78 amino acids, primarily distributed postsynaptic in the cerebral cortex, hippocampus, and olfactory bulb of human and animal brain regions. As a member of the Calpacitin protein family, Ng is a natural substrate for protein kinase C and a reservoir of calmodulin (CaM). Under physiological conditions, Ng binds to CaM to form a complex. However, under the influence of protein kinase C or oxidants, Ng can be chemically modified through phosphorylation, oxidation, and glutathioneization, reducing its affinity for CaM. This allows Ng to participate in the regulation of CaM and CaM2-activated proteases, such as CaM2-dependent NO synthase, CaM2-dependent protein kinase II (CaMKII), and CaM2-dependent adenylate cyclase. Meanwhile, since most CaM2-dependent proteases are involved in the induction of long-term potentiation (LTP) and long-term inhibition (LTD), and Ng gene expression and protein synthesis are synchronized with neuronal synapse formation and differentiation, Ng may play an important role in physiological changes such as learning, memory, and nervous system development (plasticity). Furthermore, some studies have shown that Ng may also be involved in changes in nervous system function caused by pathophysiological changes such as hypothyroidism, sleep deprivation, aging, and cerebral hypoxia preconditioning. Platelet membrane glycoproteins (GP) are components of receptors for collagen, thrombin, von Willebrand factor, fibrinogen, and adenosine diphosphate, participating in platelet adhesion, aggregation, and activation reactions, and are the initiating link in physiological hemostasis and pathological thrombosis. GP1bβ is the Ibβ subunit of glycoprotein and is related to cell attachment, cell surface receptor signal transduction, etc. However, there are currently no reports on the application of Ng and GP1bβ or their encoding genes NRGN and GP1BB in the diagnosis of amyotrophic lateral sclerosis. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a molecular diagnostic biomarker for amyotrophic lateral sclerosis (ALS).

[0006] A second object of the present invention is to provide the application of the molecular diagnostic marker or the reagent for detecting the molecular diagnostic marker.

[0007] A third objective of this invention is to provide a product for the diagnosis of amyotrophic lateral sclerosis (ALS).

[0008] The above-mentioned objective of this invention is achieved through the following technical solution:

[0009] This invention provides a molecular diagnostic biomarker for amyotrophic lateral sclerosis (ALS), comprising the NRGN and / or GP1BB genes or their encoded proteins Ng and / or GP1bβ.

[0010] The inventors discovered that peripheral leukocytes in ALS can provide clues for identifying potential biomarkers. Through high-throughput deep RNA sequencing of leukocytes and validation by qRT PCR, we identified the genes NRGN and GP1BB, which are expressed significantly differently in ALS patients and controls. Further ELISA measurements of plasma from ALS patients and controls confirmed significant differences in Ng and GP1bβ protein expression between the two groups. The area under the curve (AUC) for Ng alone in diagnosing ALS reached 80.6%, and for GP1bβ alone, it reached 71%. The combined application of GP1bβ and Ng levels showed an accuracy of 87.5% in identifying ALS patients in controls, with an AUC of 0.94. By expanding the patient sample size, we found a significant positive correlation between plasma Ng levels and ALS progression rate, particularly with the progression rate at the first symptom milestone. Ng expression was significantly correlated with ALS progression, with a PCC of 0.57 (P-value = 3.4 × 10⁻⁶). -6 In particular, Ng expression was significantly correlated with the early progression rate of ALS (PCC = 0.45 (8.8 × 10⁻⁶)). -3 The above results indicate that plasma Ng, GP1bβ, or their combination are reliable biomarkers for the diagnosis of ALS. Furthermore, plasma Ng levels are significantly positively correlated with ALS progression rate, suggesting the potential of Ng in predicting patient survival and its application in predicting ALS disease progression.

[0011] Therefore, this application seeks protection for the use of reagents for detecting the NRGN and / or GP1BB genes or their encoded proteins Ng and / or GP1bβ in the following aspects:

[0012] Application of NRGN and / or GP1BB genes or their encoded proteins Ng and / or GP1bβ as molecular diagnostic markers in the preparation of early diagnostic products for amyotrophic lateral sclerosis.

[0013] Application of the NRGN gene or its encoded protein Ng as a molecular diagnostic marker in the preparation of products for predicting the progression of amyotrophic lateral sclerosis (ALS).

[0014] Application of reagents for detecting NRGN and / or GP1BB genes or their encoded proteins Ng and / or GP1bβ in the preparation of early diagnostic products for amyotrophic lateral sclerosis (ALS).

[0015] Application of reagents for detecting the NRGN gene or its encoded protein Ng in the preparation of products for predicting the progression of amyotrophic lateral sclerosis (ALS).

[0016] Preferably, the reagents for detecting NRGN and / or GP1BB genes are PCR reagents or Southern blot reagents, including but not limited to conventional PCR, quantitative real-time PCR, fluorescent probes, etc.

[0017] Preferably, the reagent for detecting Ng and / or GP1bβ is an enzyme-linked immunosorbent assay (ELISA) reagent, and more preferably a reagent for ELISA.

[0018] Preferably, the reagent for detecting Ng and / or GP1bβ is a Western blot reagent.

[0019] Preferably, the reagent for detecting Ng and / or GP1bβ is a reagent used in protein chip detection methods.

[0020] The present invention also provides a product for early diagnosis and disease progression prediction of amyotrophic lateral sclerosis (ALS), containing reagents for detecting the NRGN and / or GP1BB genes or their encoded proteins Ng and / or GP1bβ.

[0021] Preferably, the reagent for detecting the NRGN and / or GP1BB gene or its encoded protein Ng and / or GP1bβ is the same reagent for detecting the NRGN and / or GP1BB gene or its encoded protein Ng and / or GP1bβ in human plasma leukocytes.

[0022] More preferably, the reagent for detecting NRGN and / or GP1BB genes is a PCR reagent or a Southern blot reagent, including but not limited to conventional PCR, quantitative real-time PCR, fluorescent probes, etc.

[0023] More preferably, the reagent for detecting Ng and / or GP1bβ is an enzyme-linked immunosorbent assay (ELISA) reagent, preferably a reagent for ELISA. Examples include: human Ng ELISA kits and human GP1bβ ELISA kits.

[0024] More preferably, the reagent for detecting Ng and / or GP1bβ is a Western blot reagent.

[0025] More preferably, the reagent for detecting Ng and / or GP1bβ is a reagent used in protein chip detection methods.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] This invention provides novel molecular diagnostic biomarkers for amyotrophic lateral sclerosis (ALS), Ng and / or GP1bβ. Studies show that Ng and / or GP1bβ proteins are specifically and significantly upregulated in plasma. The area under the curve (AUC) for Ng alone in diagnosing ALS reaches 80.6%, while the AUC for GP1bβ alone reaches 71%. The combined AUC for Ng and GP1bβ in diagnosing ALS reaches 94%, with a specificity of 85%. Furthermore, Ng expression is significantly correlated with the progression of ALS, with a PCC of 0.57 (P-value = 3.4 × 10⁻⁶). -6 In particular, Ng expression was significantly correlated with the early progression rate of ALS (PCC = 0.45 (8.8 × 10⁻⁶)). -3 This indicates that Ng can also serve as a molecular diagnostic marker for the rate of ALS progression, used to predict patient survival. This invention enables early diagnosis and prediction of ALS progression, and can be widely applied in the medical field for the auxiliary diagnosis and treatment of ALS. Attached Figure Description

[0028] Figure 1 To isolate and detect the expression of NRGN and GP1bβ gene expression proteins Ng and GP1bβ in leukocytes from the blood of patients with amyotrophic lateral sclerosis (ALS).

[0029] Figure 2 The diagnostic efficacy of Ng and GP1bβ expressed by the NRGN and GP1BB genes.

[0030] Figure 3 Correlation analysis of Ng with the progression of amyotrophic lateral sclerosis (ALS). Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0032] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0033] Human Ng ELISA kit (Aviva systems biology, catalog number: OKEH01858);

[0034] Human GP1bβ ELISA kit (Aviva Systems Biology, catalog number: OKEH01425).

[0035] 1. Kit composition and reagent preparation:

[0036] (1) ELISA plate: one (96 wells)

[0037] (2) Standard: 2 bottles. Dilute each bottle to 1 mL with sample diluent before use. After sealing, let stand for at least 10 minutes, then repeatedly invert / rub to aid dissolution. The concentration is 20 ng / mL. Perform serial dilutions (Note: Do not perform serial dilutions directly on the plate) to obtain concentrations of 20 ng / mL, 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.625 ng / mL, and 0.312 ng / mL. The sample diluent is used directly as the standard concentration of 0 ng / mL. Prepare within 15 minutes before use. To prepare a 10 ng / mL standard: Add 0.5 mL (not less than 0.5 mL) of the 20 ng / mL standard to an Eppendorf tube containing 0.5 mL of sample diluent and mix well. Repeat this process for other concentrations.

[0038] (3) Sample diluent: 1×20mL / bottle.

[0039] (4) Test diluent A: 1×10mL / bottle.

[0040] (5) Test diluent B: 1×10mL / bottle.

[0041] (6) Detection solution A (i.e., Neurogranin (Ng) or GP1bβ antibody): 1×120μL / vial (1:100). Dilute with detection diluent A at a ratio of 1:100 before use. Prepare the total amount required for each experiment (100μL per well) according to the pre-calculated total amount. In actual preparation, prepare 0.1-0.2mL more. For example, prepare 1μL of detection solution A with 99μL of detection diluent A, mix gently, and prepare within one hour before use.

[0042] (7) Detection solution B (i.e., secondary antibody corresponding to Neurogranin(Ng) or GP1bβ antibody): 1×120μL / vial (1:100). Dilute with detection diluent B 1:100 before use. The dilution method is the same as that for detection solution A.

[0043] (8) Substrate solution: 1×10mL / bottle.

[0044] (9) Concentrated washing solution: 1×30mL / bottle, each bottle should be diluted 25 times with distilled water before use.

[0045] (10) Termination solution: 1×10mL / bottle.

[0046] (11) Lamination: 1×5 sheets.

[0047] 2. Operating steps:

[0048] Before starting the experiment, please prepare all reagents in advance. When diluting reagents or samples, ensure thorough mixing, avoiding foaming as much as possible. A standard curve should be prepared for each test. If the sample concentration is too high, dilute it with sample diluent to bring the sample within the detection range of the kit. It is recommended that each laboratory conduct a preliminary experiment before operation to establish the optimal dilution factor.

[0049] (1) Sample addition: Set up blank wells, standard wells, and sample wells. Add 100 μL of sample diluent to the blank wells, and add 100 μL of standard or sample to the remaining wells, taking care to avoid air bubbles. Add the sample to the bottom of the wells of the ELISA plate, trying not to touch the well walls, and gently shake to mix. Cover the ELISA plate with a cap or membrane and incubate at 37°C for 120 minutes. To ensure the validity of the experimental results, please use a fresh standard solution for each experiment.

[0050] (2) Discard the liquid, shake dry, no washing required. Add 100 μL of working solution A to each well (prepare by adding 1 μL of working solution A to 99 μL of diluent A, mix gently, and prepare within one hour before use), incubate at 37°C for 60 minutes.

[0051] (3) After incubation for 60 minutes, discard the liquid in the well, spin dry, wash the plate 3 times, soaking for 1-2 minutes each time, 350μL / well, and spin dry (you can also gently tap to dry the liquid in the well).

[0052] (4) Add 100 μL of detection solution B working solution (same as detection solution A working solution) to each well, incubate at 37°C for 60 minutes.

[0053] (5) After incubation for 60 minutes, discard the liquid in the well, spin dry, wash the plate 5 times, soaking for 1-2 minutes each time, 350μL / well, and spin dry (you can also gently tap to dry the liquid in the well).

[0054] (6) Add 90 μL of substrate solution to each well in sequence and develop color at 37°C in the dark (within 30 minutes, at which point the first 3-4 wells of the standard will show a clear gradient of blue, while the gradient in the last 3-4 wells will not be obvious, at which point the process can be stopped).

[0055] (7) Add 50 μL of the stop solution to each well sequentially to terminate the reaction. The blue color will immediately turn yellow. The order of adding the stop solution should be as similar as possible to the order of adding the substrate solution. To ensure the accuracy of the experimental results, the stop solution should be added as soon as possible after the substrate reaction time has elapsed.

[0056] (8) Measure the optical density (OD value) of each well sequentially at a wavelength of 450 nm using an ELISA reader. Perform the test immediately after adding the stop solution.

[0057] Note:

[0058] (1) Leave one well as a blank zeroing well for each experiment (different from the blank well). Do not add any reagents to this well, only add the substrate solution and the stop solution at the end. Use this well to adjust the OD value to zero before measurement.

[0059] (2) Incubate strictly according to the specified time and temperature to ensure accurate results. All reagents must reach room temperature before use. Store reagents refrigerated immediately after use.

[0060] (3) Incorrect plate washing can lead to inaccurate results. Before adding detection solution B or substrate, ensure that the liquid in the wells is as dry as possible. Do not allow the microwells to dry out during incubation. To prevent sample evaporation, place the reaction plate in a sealed box lined with a damp cloth during the experiment. Cover or cover the ELISA plate. After washing and patting the plate dry, add the subsequent reagents immediately. Avoid letting the microwells dry out.

[0061] (4) Remove any residual liquid and fingerprints from the bottom of the plate, otherwise it will affect the OD value.

[0062] (5) Avoid direct exposure to strong light during storage and incubation.

[0063] (6) Unused ELISA plates or reagents should be stored at 2-8℃. Standards, working solution A, and working solution B should be prepared and used in the required quantities. Do not reuse diluted standards, working solution A, or working solution B.

[0064] (7) It is recommended to use two wells for each sample test to ensure the accuracy of the test results.

[0065] 3. Board washing method

[0066] Manual plate washing method: Aspirate (without touching the plate wall) or shake off the liquid in the microplate; place several layers of absorbent paper on the lab bench, and tap the microplate face down several times; inject at least 0.3 mL of the recommended washing buffer into the wells, soak for 1-2 minutes, and repeat this process several times as needed.

[0067] Automatic plate washing: If an automatic plate washing machine is available, it should be used in the formal experimental process only after you have become proficient in its use.

[0068] 4. Calculation

[0069] Plot a standard curve on semi-logarithmic graph paper with the concentration of the standard as the x-axis (logarithmic scale) and the OD value as the y-axis (ordinary scale). (It is recommended to use professional curve creation software, such as Curve Expert 1.3, for analysis.) Find the corresponding concentration of the sample from the standard curve based on its OD value, and then multiply it by the dilution factor. Alternatively, calculate the regression equation of the standard curve using the concentration and OD value of the standard, substitute the OD value of the sample into the equation to calculate the sample concentration, and then multiply it by the dilution factor to obtain the actual concentration of the sample.

[0070] Example 1: Validation of characteristic Neurogranin (Ng) and GP1bβ in amyotrophic lateral sclerosis (ALS)

[0071] Our team, through extensive preliminary research, discovered that Neurogranin (Ng) and GP1bβ are associated with the diagnosis of amyotrophic lateral sclerosis (ALS) and can serve as blood molecular diagnostic markers for ALS. The following section describes the use of ELISA to detect Neurogranin (Ng) and GP1bβ in blood samples from ALS patients and healthy individuals, thereby validating the characteristic Neurogranin (Ng) and GP1bβ markers for ALS.

[0072] The specific steps include: coating microplates with purified antibodies to form a solid-phase carrier; sequentially adding samples or standards, biotinylated anti-Neurogranin(Ng) and GP1bβ antibodies, and HRP-labeled avidin to microplates coated with anti-Neurogranin(Ng) and GP1bβ antibodies, respectively; after thorough washing, developing the color with the substrate TMB. TMB is converted to blue under the catalysis of peroxidase, and then to yellow under acidic conditions. The color intensity is positively correlated with the levels of Neurogranin(Ng) and GP1bβ in the sample. The absorbance (OD value) is measured at 450 nm using a microplate reader to calculate the sample concentration.

[0073] Using blood Neurogranin (Ng) and GP1bβ to differentiate between amyotrophic lateral sclerosis (ALS) patients and healthy individuals: The expression levels of Neurogranin (Ng) and GP1bβ in the blood of ALS patients and healthy individuals were analyzed using the methods described above. The results showed that Neurogranin (Ng) and GP1bβ could be detected in the blood of ALS patients, and the levels in ALS patients were higher than those in healthy individuals. Figure 1 The area under the curve (AUC) for the accuracy of Ng alone in diagnosing ALS reached 80.6%, and the AUC for GP1bβ alone in diagnosing ALS reached 71%. The combined accuracy of Neurogranin (Ng) and GP1bβ in diagnosing ALS reached 94%, with a specificity of 85%. Figure 2 ).

[0074] Example 2: Neurogranin (Ng) Kit for Predicting the Course of Amyotrophic Lateral Sclerosis

[0075] Our team, through extensive preliminary research, discovered that the expression of Neurogranin (Ng) in the blood is significantly correlated with the progression of amyotrophic lateral sclerosis (ALS), particularly with the early progression rate of ALS. Therefore, it can serve as a blood molecular diagnostic marker for the disease progression, especially in the early stages of ALS. The following describes an ELISA assay using blood samples from ALS patients and healthy individuals to detect Neurogranin (Ng) and predict the course of ALS, particularly its early onset.

[0076] The ELISA test was the same as in Example 1.

[0077] We measured neurogranin (Ng) levels in the plasma of 59 patients using ELISA. Neurogranin (Ng) levels increased with increasing progression of amyotrophic lateral sclerosis (ALS). Figure 3 We found a significant correlation between Ng levels and ALS progression rate, with a Pearson correlation coefficient (PCC) of 0.57 (P-value = 3.4 × 10⁻⁶). -6 Furthermore, we detected a significant correlation between plasma neurogranin (Ng) levels and the early progression rate of ALS in patients, with Ng expression being significantly correlated with the early progression rate of ALS (PCC = 0.45 (8.8 × 10⁻⁶)). -3 Furthermore, there is a correlation between neurogranin (Ng) levels and the progression rate of amyotrophic lateral sclerosis (ALS) in patients at different stages. The progression rate of ALS can distinguish between patients who survive for 1 or 2 years after diagnosis and those who survive for 6 years or more. Therefore, plasma Ng can also predict early survival in ALS patients and can be used to predict disease progression.

[0078] The above embodiments are preferred implementations and some application examples of the present invention. However, the use of the present invention is not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. Application of a reagent for detecting GP1bβ protein in the preparation of early diagnostic products for amyotrophic lateral sclerosis (ALS); the reagent is a reagent for detecting protein levels.

2. Application of reagents for detecting neurogranulin proteins Ng and GP1bβ in the preparation of early diagnostic products for amyotrophic lateral sclerosis (ALS); the reagents are for detecting protein levels.

3. The application according to claim 1 or 2, characterized in that, The reagent used to detect protein levels is an enzyme-linked immunosorbent assay (ELISA) reagent.

4. The application according to claim 1 or 2, characterized in that, The reagent used to detect protein levels is a Western blot reagent.

5. The application according to claim 1 or 2, characterized in that, The reagents used to detect protein levels are those used in protein chip detection methods.

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