Application of plasma neurofilament light chain protein combined with ceramide in diagnosis of progressive supranuclear palsy

Abstract

This invention discloses the application of plasma neurofilament light chain protein combined with ceramides in the diagnosis of progressive supranuclear palsy (PSP), relating to the field of biomedical technology. This invention provides biomarkers for diagnosing PSP, including a combination of ceramides or a combination of plasma neurofilament light chain protein combined with ceramides; the ceramide combination includes Cer 16, Cer 18, Cer 24:0, Cer 24:1, Cer 16 / Cer 24:0, Cer 18 / Cer 24:0, and Cer 24:1 / Cer 24:0. This invention develops a plasma ceramide combination as a biomarker for diagnosing PSP, and by measuring the concentration of NfL in plasma with a specific ceramide combination and substituting it into a specific logistic regression calculation formula, it solves the problem of insufficient efficacy of current biomarkers in diagnosing PSP, significantly improving the diagnostic accuracy of PSP.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of plasma neurofilament light chain protein combined with ceramide in the diagnosis of progressive supranuclear palsy. Background Technology

[0002] Progressive supranuclear palsy (PSP) is a rare and fatal neurodegenerative disease (belonging to the Tau protein disease) with highly insidious early clinical symptoms. Currently, the clinical diagnosis of PSP relies heavily on the appearance of core motor symptoms and imaging features (such as the "hummingbird sign" on MRI). By the time these features are clear, patients are often already in the middle or late stages of the disease, missing the window for early intervention.

[0003] In the field of blood biomarkers, neurofilament light chain protein (NfL) is considered a universal indicator of axonal damage in the central nervous system, and it is elevated to some extent in the peripheral blood of PSP patients. On the other hand, abnormal lipid metabolism (especially ceramide-mediated changes in cell membrane structure and apoptosis signaling) plays a crucial role in neurodegenerative diseases. Current research explores changes in blood ceramides in neurodegenerative diseases.

[0004] Despite the progress made in the research of biomarkers for neurodegenerative diseases, there are still problems and limitations: 1. Existing biomarkers lack disease specificity and have limited efficacy: a single increase in NfL can only indicate nerve axon damage and is not specific to PSP; 2. There is a lack of multidimensional differential diagnostic models.

[0005] Therefore, developing new biomarkers and establishing efficient and accurate diagnostic models are of great significance for the diagnosis of progressive supranuclear palsy. Summary of the Invention

[0006] The purpose of this invention is to provide the application of plasma neurofilament light chain protein combined with ceramides in the diagnosis of progressive supranuclear palsy (PSP), thereby addressing the problems existing in the prior art. This invention develops a combination of plasma ceramides as a biomarker for diagnosing PSP. By measuring the concentration of NfL in plasma in combination with a specific ceramide and incorporating it into a specific logistic regression calculation formula, it solves the problem of insufficient efficacy of current biomarkers in diagnosing PSP, significantly improving the diagnostic accuracy of PSP and possessing clinical application value.

[0007] To achieve the above objectives, the present invention provides the following solution: This invention provides a biomarker for diagnosing progressive supranuclear palsy, wherein the biomarker is a combination of ceramides; The ceramide combination includes Cer 16, Cer 18, Cer 24:0, Cer 24:1, Cer 16 / Cer 24:0, Cer 18 / Cer 24:0 and Cer 24:1 / Cer 24:0.

[0008] The present invention also provides the use of reagents for detecting the above-mentioned biomarkers in the preparation of products for diagnosing progressive supranuclear palsy.

[0009] The present invention also provides the application of the above-mentioned biomarkers in constructing a diagnostic model for progressive supranuclear palsy.

[0010] The present invention also provides a combination of biomarkers for diagnosing progressive supranuclear palsy, wherein the combination of biomarkers is a combination of plasma neurofilament light chain protein and ceramide; The ceramide combination includes Cer 16, Cer 18, Cer 24:0, Cer 24:1, Cer 16 / Cer 24:0, Cer 18 / Cer 24:0 and Cer 24:1 / Cer 24:0.

[0011] The present invention also provides the use of reagents for detecting the above-mentioned combination of biomarkers in the preparation of products for diagnosing progressive supranuclear palsy.

[0012] The present invention also provides the application of the above-mentioned combination of biomarkers in constructing a diagnostic model for progressive supranuclear palsy.

[0013] This invention also provides a method for constructing a diagnostic model for progressive supranuclear palsy, comprising the following steps: the diagnostic model is constructed using plasma concentrations of Cer 16, Cer 18, Cer 24:0, Cer 24:1, Cer16 / Cer 24:0 ratio, Cer 18 / Cer 24:0 ratio, and Cer 24:1 / Cer 24:0 ratio as input variables; or it is constructed using plasma concentrations of neurofilament light chain protein, Cer 16, Cer 18, Cer 24:0, Cer 24:1, Cer 16 / Cer 24:0 ratio, Cer 18 / Cer 24:0 ratio, and Cer 24:1 / Cer 24:0 ratio as input variables; The predicted probability value of the diagnostic model is P=1 / (1+e^-Y); Among them, Y= 0.2320 - 0.0473 × [Cer 16] + 0.3266 × [Cer 18] - 0.0004 × [Cer 24:0] - 0.0253 × [Cer 24:1] + 0.0206 × [Cer 16 / Cer 24:0] + 0.0114 × [Cer 18 / Cer 24:0]+ 0.0911 × [Cer 24:1 / Cer 24:0]; Or, Y = -39.5970 + 0.6680 × [NfL] - 0.1548 × [Cer 16] + 1.1084 × [Cer 18] + 0.0003 × [Cer 24:0] - 0.0400 × [Cer 24:1] + 0.0106 × [Cer 16 / Cer24:0] + 0.0029 × [Cer 18 / Cer 24:0] + 0.0192 × [Cer 24:1 / Cer 24:0].

[0014] The present invention also provides a diagnostic model for progressive supranuclear palsy constructed by the above-described method.

[0015] The present invention discloses the following technical effects: This invention has discovered that by detecting the concentration of specific ceramide combinations (Cer 16, Cer 18, Cer 24:0, Cer 24:1, Cer 16 / Cer 24:0, Cer 18 / Cer 24:0 and Cer 24:1 / Cer 24:0) in plasma, PSP can be accurately diagnosed, providing a highly efficient diagnostic biomarker.

[0016] This invention also, for the first time, transcends different pathological mechanisms (NfL represents nerve axonal injury, and the ceramide combination represents systemic lipid metabolism disorder) by constructing a multivariate logistic regression model using a specific ceramide combination and NfL. By measuring the concentrations of NfL and specific ceramides in plasma and substituting them into a specific logistic regression calculation formula, it solves the problems of insufficient efficacy and cumbersome operation of current biomarkers in diagnosing PSP. In diagnosing PSP, the diagnostic AUC of NfL alone was 0.958, and the diagnostic AUC of the combination ceramide alone was 0.986; while the combined diagnostic model successfully improved the AUC to 1.0000, achieving absolute differentiation of 100.0% sensitivity and 100.0% specificity. This method requires only a very small amount of peripheral plasma, is convenient to detect, and has extremely high clinical translation and commercial application value. Attached Figure Description

[0017] 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.

[0018] Figure 1 Comparison of ROC curves for differentiating PSP from HC in diagnostic models of plasma NfL and / or ceramides. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Terminology Explanation: Neurofilament light chain protein (NfL) is a key structural protein in the neuronal cytoskeleton. It is an important subunit of the neurofilament protein (NF) family and is highly expressed in myelinated axons.

[0025] Ceramides are core sphingolipid molecules in plasma containing long-chain fatty acids. They are composed of sphingosine and fatty acids linked by amide bonds and participate in cell membrane formation, apoptosis, and signal transduction. Abnormal levels of ceramides are closely related to metabolic diseases, cardiovascular diseases, and neurodegenerative diseases.

[0026] Cer 16, or ceramide subtype containing a 16-carbon saturated fatty acid chain, is mainly distributed in plasma lipoproteins and can regulate lipid metabolism and inflammatory responses. Its elevation is associated with insulin resistance and the occurrence and development of atherosclerosis.

[0027] Cer 18 refers to a ceramide subtype with 18 carbon atoms in its fatty acid chain. It is widely present in human tissues and plasma, participates in the regulation of cell proliferation and apoptosis, and abnormal expression is associated with an increased risk of obesity, type 2 diabetes, and cardiovascular disease.

[0028] Cer 24:0 is a ceramide subtype containing a 24-carbon saturated fatty acid chain. It is a ceramide component with a high content in plasma and participates in maintaining cell membrane homeostasis. Its elevated level is positively correlated with the risk of cardiovascular events such as coronary heart disease and myocardial infarction.

[0029] Cer 24:1, a ceramide subtype containing a 24-carbon monounsaturated fatty acid chain, is present in plasma and various tissues. It participates in lipid metabolism and inflammation regulation, and abnormally elevated levels can promote vascular endothelial damage and are associated with the progression of metabolic syndrome.

[0030] Previous research by the inventors' research group revealed significant differences in plasma neurofilament light chain protein (NfL) concentration and plasma ceramide concentrations between patients with progressive supranuclear palsy and healthy individuals. Based on this, the present invention conducts research on the following embodiments.

[0031] Example 1 I. Materials and Methods Sample Source: The clinical samples involved in this study were all obtained from inpatients in the Department of Movement Disorders and healthy individuals in the Physical Examination Center of Beijing Tiantan Hospital, Capital Medical University, from January 2023 to October 2025. This study was reviewed and approved by the relevant hospital's clinical research ethics committee. All sample acquisition procedures complied with the Declaration of Helsinki and relevant national regulations, and all participants signed written informed consent forms.

[0032] Subjects included 40 clinically diagnosed patients with progressive supranuclear palsy (PSP) and 40 healthy individuals (HC).

[0033] Plasma sample collection and preparation: Fasting venous blood was collected. Blood samples were collected in test tubes coated with ethylenediaminetetraacetic acid (EDTA) and immediately centrifuged at 3000 rpm for 15 minutes at 4°C. The separated supernatant plasma was transferred aliquoted to cryovials and stored at -80°C until analysis to prevent lipid degradation.

[0034] Extraction and quantification of plasma ceramides: Plasma samples were thawed on ice, and standardized volumes of each sample were mixed with an organic solvent containing a stable isotope-labeled internal standard for lipid extraction. Subsequently, the plasma ceramide profiles (including Cer 16, Cer 18, Cer24:0, Cer 24:1, etc.) were accurately quantified using a multi-batch liquid chromatography-tandem mass spectrometry (LC-MS / MS) system.

[0035] Plasma neurofilament light chain protein (NfL) detection: As a benchmark for comparison and co-modeling, the concentration of NfL in plasma was measured using photo-induced chemiluminescence immunoassay (LiCA®, Chemclin Diagnostics, Beijing, China). This method utilizes antibody-conjugated luminescent microspheres and photosensitive microspheres. When the antigen (NfL) binds and brings the microspheres close together, they are excited and generate a signal, thereby achieving highly sensitive and accurate quantification.

[0036] II. Data Processing Statistical analysis and logistic regression modeling were performed using Python software. The specific processing and analysis steps are as follows: 1. Feature Selection and Model Construction: Plasma NfL concentration and seven core lipid features (Cer16, Cer 18, Cer 24:0, Cer 24:1 concentrations and their derived ratios Cer 16 / Cer 24:0, Cer 18 / Cer 24:0, Cer 24:1 / Cer 24:0) were extracted from the subjects as predictive variables. The detection results of each biomarker in the subject population are shown in Table 1.

[0037] Table 1. Distribution of levels and ratios of various biomarkers in different subjects (mean ± standard deviation) 2. Comparison Model Specification: To verify the gain effect of the combined diagnosis, three logistic regression classification models were constructed for each differential diagnosis group: (1) Single NfL model (only NfL concentration is input); (2) Single ceramide model (only 7 ceramide features are input); (3) NfL combined with ceramide model (NfL and 7 ceramide features are input simultaneously for fitting and prediction).

[0038] 3. Algorithm and Formula Generation: The multivariate logistic regression algorithm is used for fitting, and the prediction probability formula P=1 / (1+e^-Y) is generated.

[0039] 4. Diagnostic efficacy assessment and cutoff value determination: After obtaining the predicted probability P-values ​​output by the models, receiver operating characteristic (ROC) curve analysis was used to evaluate the diagnostic discrimination ability of each model, and the area under the curve (AUC) was calculated. Subsequently, Youden's Index (which finds the maximum value of "sensitivity + specificity - 1") was used to determine the optimal diagnostic cutoff value for each combined model, and the percentages of sensitivity and specificity at this optimal cutoff value were output.

[0040] III. Results and Analysis 1. Substituting the data into the model, a formula for calculating the linear predictor variable Y, used to distinguish between PSP and HC, was established (numerical values ​​are rounded to four decimal places): Y = -5.5530 + 0.1596 × [NfL]; Y= 0.2320 - 0.0473 × [Cer 16] + 0.3266 × [Cer 18] - 0.0004 × [Cer24:0] - 0.0253 × [Cer 24:1]+ 0.0206 × [Cer 16 / Cer 24:0] + 0.0114 × [Cer18 / Cer 24:0]+ 0.0911 × [Cer 24:1 / Cer 24:0]; Y = -39.5970 + 0.6680 × [NfL] - 0.1548 × [Cer 16]+ 1.1084 × [Cer18] + 0.0003 × [Cer 24:0]- 0.0400 × [Cer 24:1] + 0.0106 × [Cer 16 / Cer 24:0]+ 0.0029 × [Cer 18 / Cer 24:0] + 0.0192 × [Cer 24:1 / Cer 24:0].

[0041] Substituting Y into the prediction probability formula to calculate the prediction probability P value, the diagnostic efficacy is tested, and the results are as follows: Figure 1As shown, the diagnostic AUC was 0.958 when plasma NfL was used alone, and 0.986 when the ceramide combination was used alone. Using the combined diagnostic model, the diagnostic AUC reached 1.000.

[0042] The optimal diagnostic cutoff value for the ceramide combination diagnostic model is 0.8596, meaning that a positive result (suspected PSP) is defined as a result when the predicted probability (P) of a subject is greater than 0.8596. At this cutoff value, the model exhibits a sensitivity of 87.5% and a specificity of 100%.

[0043] The optimal diagnostic cutoff value for the combined diagnostic model is 0.8215, meaning that a positive result (suspected PSP) is defined as a result when the predicted probability P of a subject is greater than 0.8215. At this cutoff value, the model achieves a sensitivity of 100.0% and a specificity of 100.0%.

[0044] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A biomarker for diagnosing progressive supranuclear palsy, characterized in that, The biomarker is a combination of ceramides; The ceramide combination includes Cer 16, Cer 18, Cer 24:0, Cer 24:1, Cer 16 / Cer 24:0, Cer18 / Cer 24:0 and Cer 24:1 / Cer 24:

0.

2. The use of the reagent for detecting the biomarker of claim 1 in the preparation of products for diagnosing progressive supranuclear palsy.

3. The application of the biomarker of claim 1 in constructing a diagnostic model for progressive supranuclear palsy.

4. A combination of biomarkers for diagnosing progressive supranuclear palsy, characterized in that, The biomarker combination is a combination of plasma neurofilament light chain protein and ceramide; The ceramide combination includes Cer 16, Cer 18, Cer 24:0, Cer 24:1, Cer 16 / Cer 24:0, Cer18 / Cer 24:0 and Cer 24:1 / Cer 24:

0.

5. The use of a reagent for detecting the combination of biomarkers according to claim 4 in the preparation of a product for diagnosing progressive supranuclear palsy.

6. The application of the biomarker combination of claim 4 in constructing a diagnostic model for progressive supranuclear palsy.

7. A method for constructing a diagnostic model for progressive supranuclear palsy, characterized in that, Includes the following steps: The diagnostic model was constructed using plasma Cer 16 concentration, Cer 18 concentration, Cer 24:0 concentration, Cer 24:1 concentration, Cer 16 / Cer24:0 ratio, Cer 18 / Cer 24:0 ratio, and Cer 24:1 / Cer 24:0 ratio as input variables; or it was constructed using plasma neurofilament light chain protein concentration, Cer 16 concentration, Cer 18 concentration, Cer 24:0 concentration, Cer 24:1 concentration, Cer16 / Cer 24:0 ratio, Cer 18 / Cer 24:0 ratio, and Cer 24:1 / Cer 24:0 ratio as input variables. The predicted probability value of the diagnostic model is P=1 / (1+e^-Y); Among them, Y= 0.2320 - 0.0473 × [Cer 16] + 0.3266 × [Cer 18] - 0.0004 × [Cer24:0] - 0.0253 × [Cer 24:1] + 0.0206 × [Cer 16 / Cer 24:0] + 0.0114 × [Cer18 / Cer 24:0] + 0.0911 × [Cer 24:1 / Cer 24:0]; Or, Y = -39.5970 + 0.6680 × [NfL] - 0.1548 × [Cer 16] + 1.1084 × [Cer18] + 0.0003 × [Cer 24:0] - 0.0400 × [Cer 24:1] + 0.0106 × [Cer 16 / Cer 24:0] + 0.0029 × [Cer 18 / Cer 24:0] + 0.0192 × [Cer 24:1 / Cer 24:0].

8. A diagnostic model for progressive supranuclear palsy constructed by the construction method of claim 7.

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