Biomarker for predicting or diagnosing motor complications of Parkinson's disease and application of biomarker

Abstract

The invention discloses a biomarker for predicting or diagnosing motor complications of Parkinson's disease and application of the biomarker, and belongs to the technical field of biological medicine, the invention provides the biomarker for predicting or diagnosing the motor complications of Parkinson's disease by taking the plasma NfL level as the biomarker for predicting or diagnosing the motor complications of Parkinson's disease for the first time, and the biomarker can be obtained by comparing the baseline plasma NfL, GFAP, A beta 40, A beta 42 and P-tau181 levels. The increase of the baseline plasma NfL level is significantly related to the occurrence risk of motor complications (including motion fluctuation and dyskinesia) of Parkinson's disease patients, and a basis can be provided for clinical early intervention.

Description

Technical Field

[0001] The present invention relates to a biomarker for predicting or diagnosing motor complications of Parkinson's disease and its application, specifically to the application of a biomarker NfL for predicting or diagnosing motor complications of Parkinson's disease and a substance for detecting the biomarker NfL in the preparation of products for predicting or diagnosing motor complications of Parkinson's disease, belonging to the field of biomedical technology. Background Art

[0002] Parkinson's disease (PD) is a common neurodegenerative disease, mainly characterized by the degeneration of dopaminergic neurons in the substantia nigra-striatum and the formation of Lewy bodies. Anti-PD drugs (especially levodopa) play an important role throughout the disease process. However, with the progression of the disease and the increase in levodopa dosage, patients often experience motor complications such as motor fluctuations (54.3%-100%) and dyskinesia (14.5%-55.7%). These complications not only seriously affect the quality of life of patients, but may even lead to disability. Therefore, early prediction and effective intervention of motor complications are crucial for PD patients.

[0003] Previous studies have found a variety of clinical features and non-motor symptoms that can predict motor complications, including age of onset, disease duration, anxiety, and apathy. Neuroimaging studies have shown that changes in white matter connections in the cortico-striatal region and abnormal brain functional connections are related to motor complications. Positron emission tomography studies have suggested that a lower dopamine transporter binding capacity in early PD patients may predict the occurrence of motor complications. However, the subjectivity of clinical scales and the high requirements of neuroimaging techniques limit their clinical application efficiency. Therefore, it is of great significance to develop reliable and convenient biomarkers.

[0004] Neurofilament light chain protein (NfL), as a biomarker for axonal injury, is one of the most effective blood biomarkers for predicting the progression of PD disease. Glial fibrillary acidic protein (GFAP) is a biomarker for reactive astrocytes and neuroinflammation, and is related to cognitive decline and disease progression in PD, Alzheimer's disease (AD), and frontotemporal dementia. Phosphorylated tau181 (p-tau181), as a tau pathology biomarker, and amyloid-β 42 (Aβ42) and 40 (Aβ40), as amyloid plaque deposition biomarkers, can be used to detect the co-pathology of AD in PD (especially dementia with Lewy bodies). However, there is currently no study to clarify whether these plasma biomarkers are related to PD motor complications, nor is it clear whether they can predict the occurrence of motor complications. Summary of the Invention

[0005] The present invention aims to solve the existing gap in biomarkers related to predicting PD motor complications, and provides a biomarker for predicting or diagnosing PD motor complications and its applications. For the first time, it is proposed to use the blood NfL level as a biomarker, which has extremely high accuracy, specificity and sensitivity for the prediction or diagnosis of PD motor complications.

[0006] The present invention is achieved through the following technical solutions: A biomarker for predicting or diagnosing PD motor complications, by detecting the NfL level content in the blood sample of PD patients, to predict or diagnose whether PD patients have motor complications.

[0007] The application of substances for detecting the NfL level in the preparation of products for predicting or diagnosing PD motor complications, for the blood samples of PD patients, to check the content of their NfL level.

[0008] The blood sample includes plasma.

[0009] The detection method includes single molecule array Simoa detection technology.

[0010] The substances include kits, detection reagents, test strips or other substances that can be achieved in medicine.

[0011] When PD patients initially have no motor fluctuations, when the detected NfL level is greater than 13.66 pg / mL, it is predicted or diagnosed that the probability of the PD patient transforming from initially having no motor fluctuations to having motor fluctuations is relatively high; when PD patients initially have no dyskinesia, when the detected NfL level is greater than 13.74 pg / mL, it is predicted or diagnosed that the probability of the PD patient transforming from initially having no dyskinesia to having dyskinesia is relatively high.

[0012] It should be noted that in the present invention, "no motor fluctuations" means that PD patients have no motor fluctuations at baseline, "transforming into motor fluctuations" means that in subsequent follow-up, PD patients have motor fluctuations, "no dyskinesia" means that PD patients have no dyskinesia at baseline, and "transforming into dyskinesia" means that in subsequent follow-up, PD patients have dyskinesia.

[0013] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0014] (1) The present invention for the first time proposes to use the plasma NfL level as a biomarker for predicting or diagnosing PD patients' motor complications, and it can be obtained by comparing the baseline plasma NfL, GFAP, Aβ40, Aβ42 and P-tau181 levels. An increase in the baseline plasma NfL level is significantly associated with the occurrence risk of PD patients' motor complications (including motor fluctuations and dyskinesia), which can provide a basis for early clinical intervention.

[0015] (2) The present invention can be demonstrated through a large number of clinical samples and follow-up verifications that using plasma NfL levels to predict motor complications (including motor fluctuations and dyskinesia) has extremely high accuracy, specificity, and sensitivity.

[0016] (3) The present invention can use the ultrasensitive single molecule array Simoa detection technology to detect plasma neurofilament light chain protein in Parkinson's disease patients, thereby obtaining plasma NfL levels, which can be quickly and efficiently applied to clinical diagnosis to improve detection efficiency. Brief Description of the Drawings

[0017] Figure 1 It is the baseline plasma biomarker level in PD patients with motor complications after 7 years of follow-up.

[0018] Figure 2 It is the Kaplan-Meier survival curve of the baseline plasma NfL concentration and the occurrence of motor complications.

[0019] Figure 3 It is the ROC curve of plasma NfL concentration predicting the occurrence of motor complications. Detailed Description of the Invention

[0020] The invention object, technical solution, and beneficial effects of the present invention will be further described in detail below.

[0021] It should be noted that the following detailed descriptions are all exemplary and are intended to provide further explanations for the claimed present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0022] Parkinson's disease (PD) is a common neurodegenerative disease, mainly characterized by the degeneration of substantia nigra-striatal dopaminergic neurons and the formation of Lewy bodies. Anti-PD drugs (especially levodopa) play an important role throughout the disease process. However, as the disease progresses and the levodopa dose increases, patients often experience motor complications such as motor fluctuations (54.3%-100%) and dyskinesia (14.5%-55.7%). These complications not only seriously affect the quality of life of patients but may even lead to disability. Therefore, early prediction and effective intervention of motor complications are crucial for PD patients, but currently, no biomarker has been found that can predict or diagnose PD motor complications.

[0023] Neurofilament light chain protein is considered a marker of axonal degeneration. Existing literature has reported that elevated serum NfL levels in early Parkinson's disease are associated with cognitive and motor impairments (see "Serum neurofilament light at diagnosis: a prognostic indicator for accelerated disease progression in Parkinson’s Disease", npj Parkinson's Disease, 2024); and that serum NfL levels are related to the severity and progression of motor decline (see "Neurofilament light as a biomarker for motor decline in Parkinson’s disease", Frontiers in Neuroscience, 2022). However, no correlation has been found between NfL levels and the prediction of the occurrence of motor complications in PD. Motor complications, including motor fluctuations and dyskinesia, refer to the emergence of fluctuations in treatment efficacy and involuntary limb movements, which are significantly different from motor impairments and motor decline, both of which refer to a decrease in motor ability.

[0024] The present invention for the first time uses NfL as a biomarker to predict or diagnose motor complications in Parkinson's disease. By taking the blood NfL level as the detection index, the occurrence of motor complications in Parkinson's disease patients is predicted or diagnosed by detecting the NfL level content in the blood samples of Parkinson's disease patients, with extremely high accuracy, specificity, and sensitivity.

[0025] The present invention will be further described in detail below in conjunction with embodiments, but the implementation manners of the present invention are not limited thereto.

[0026] Example 1:

[0027] This example relates to a product for predicting or diagnosing motor complications in Parkinson's disease, which is a kit for detecting the NfL level content in the blood samples of Parkinson's disease patients.

[0028] Specifically, a high-sensitivity single-molecule detection platform simoa is used to detect plasma NfL samples, and the detection work is carried out on an HD-X instrument according to the kit instructions.

[0029] (1) The samples are thawed at room temperature, shaken well, and centrifuged at low temperature at 12,000 rpm for 5 min at 4°C.

[0030] The standard products are configured in the kit; the samples are from the plasma of Parkinson's disease patients; the sample inclusion standard is no repeated freezing and thawing.

[0031] (2) Add the corresponding volume of standard products to the 96-well plate and load the samples.

[0032] (3) Load the corresponding consumables, basic buffer solution and reagents, confirm the program, and start running the detection program. The detection program is as follows:

[0033] Wash the plate 3 times with 1X MSD Wash Buffer or PBS-T, 150 μL per well; add 50 μL of coating solution to each well, seal the plate, and incubate on a shaker at room temperature (22°C - 27°C) at 700 rpm for 1 hour; wash the plate 3 times, add 25 μL of blocking solution to each well, and add 25 μL of calibrator or sample (after dilution) to each well; seal the plate and incubate on a shaker at room temperature for 1.5 hours (maintaining 22°C - 27°C).

[0034] Prepare the TURBO-BOOST antibody solution (single detection). 6 mL of working solution is required for each plate. Vortex thoroughly; then add the TURBO-BOOST antibody, wash the plate 3 times, add 50 μL of antibody solution to each well, seal the plate, and incubate on a shaker at room temperature for 1 hour (maintaining 22°C - 27°C); prepare the enhancement solution (Enhance Solution) by mixing in proportion: 2970 μL of molecular-grade water + 1500 μL of Enhance E1 (4X) + 1500 μL of Enhance E2 (4X) + 30 μL of Enhance E3 (200X). Vortex thoroughly; add the enhancement solution, wash the plate 3 times, add 50 μL of enhancement solution to each well, seal the plate, and incubate on a shaker at room temperature for 30 minutes (maintaining 22°C - 27°C).

[0035] Prepare the TURBO-TAG detection solution (Detect Solution) by mixing in proportion: 4470 μL of molecular-grade water + 1500 μL of Detect D1 (4X) + 30 μL of Detect D2 (200X). Store in the dark and vortex thoroughly; add the detection solution, wash the plate 3 times, add 50 μL of detection solution to each well, seal the plate, and incubate on a shaker at 27°C for 1 hour (temperature control equipment is required); finally, wash the plate and read the data. Wash the plate 3 times (low-speed washing program to avoid residual bubbles), and add 150 μL of MSD GOLD Read Buffer B to each well. Immediately read the signal using an MSD instrument (such as QuickPlex SQ 120).

[0036] (4) Obtain the NfL level content of the sample.

[0037] Example 2:

[0038] This example involves using the Cox regression model to analyze and explore the factors affecting the occurrence of motor complications in Parkinson's disease patients.

[0039] First, a univariate Cox regression analysis was used to explore the influencing factors for the occurrence of motor complications. The univariate Cox regression analysis showed that motor fluctuations were related to the daily dose of levodopa at baseline, anxiety, depression, plasma NfL concentration, and plasma GFAP concentration; dyskinesia was related to the disease duration at baseline, daily dose of levodopa, UPDRS-I, UPDRS-II, UPDRS-III, anxiety, depression, plasma NfL concentration, and plasma GFAP concentration.

[0040] Then, a multivariate Cox regression analysis was used to adjust for the daily dose of levodopa, anxiety, depression, plasma NfL concentration, and plasma GFAP concentration. The results showed that a higher plasma NfL at baseline was the main factor affecting the occurrence of motor fluctuations; a multivariate Cox regression analysis was used to adjust for the disease duration, daily dose of levodopa, UPDRS-I, UPDRS-II, UPDRS-III, anxiety, depression, plasma NfL concentration, and plasma GFAP concentration. The results showed that a higher plasma NfL at baseline was the main factor affecting the occurrence of dyskinesia.

[0041] See Tables 1 and 2 below for details.

[0042] Table 1: Cox regression analysis for predicting motor fluctuations

[0043]

[0044]

[0045] Table 2: Cox regression analysis for predicting dyskinesia

[0046]

[0047]

[0048] Example 3:

[0049] This example involves the clinical verification and follow-up of the occurrence of motor complications in PD patients.

[0050] A total of 173 early PD patients were enrolled in this study, and the plasma NfL concentrations of 173 patients at baseline, 1 year, and 2 years were detected using high-sensitivity single-molecule detection. 173 PD patients completed the 1-year follow-up, 167 completed the 2-year follow-up, 147 completed the 3-year follow-up, and 123 completed the 4-year follow-up. Among them, 77 completed the 5-year follow-up, 30 completed the 6-year follow-up, and 8 completed the 7-year follow-up.

[0051] Table 3: Baseline Clinical Characteristics of PD Patients with Motor Fluctuations and Dyskinesia

[0052]

[0053]

[0054] Table 3 above shows the occurrence of motor complications in PD patients after 7 years of follow-up. Among them, 173 PD patients were enrolled at baseline, and 72 patients had motor fluctuations at the end of the 7-year follow-up. The incidence of motor fluctuations was 41.62%. At the end of the 7-year follow-up, 31 PD patients had dyskinesia, and the incidence of dyskinesia was 17.91%.

[0055] Figure 1 Baseline plasma biomarker levels in PD patients during motor complications after 7 years of follow-up. Figure 1 In it, A is the baseline NfL level of patients with motor fluctuations after 7 years of follow-up; Figure 1 In it, B is the baseline GFAP level of patients with motor fluctuations after 7 years of follow-up; Figure 1 In it, C is the baseline Aβ40 level of patients with motor fluctuations after 7 years of follow-up; Figure 1 In it, D is the baseline Aβ42 level of patients with motor fluctuations after 7 years of follow-up; Figure 1 In it, E is the baseline P-tau181 level of patients with motor fluctuations after 7 years of follow-up; Figure 1 In it, F is the baseline NfL level of patients with dyskinetic symptoms after 7 years of follow-up; Figure 1 In it, G is the baseline GFAP level of patients with dyskinetic symptoms after 7 years of follow-up; Figure 1 In it, H is the baseline Aβ40 level of patients with dyskinetic symptoms after 7 years of follow-up; Figure 1 In it, I is the baseline Aβ42 level of patients with dyskinetic symptoms after 7 years of follow-up; Figure 1 In it, J is the baseline P-tau181 level of patients with dyskinetic symptoms after 7 years of follow-up. It can be seen from Figure 1 that PD patients with motor fluctuations and dyskinesia during follow-up have higher baseline NfL.

[0056] Figure 2 Kaplan-Meier survival curve of baseline plasma NfL concentration and occurrence of motor complications Figure 2 In it, A represents the baseline plasma NfL tertile. The highest baseline NfL group has a higher incidence of motor fluctuations than the lowest group. Figure 2 In it, B represents the baseline plasma NfL tertile. The highest baseline NfL group has a higher incidence of dyskinesia than the lowest group. It can be seen from Figure 2 that PD patients with higher baseline NfL are more likely to have motor fluctuations and dyskinesia.

[0057] Figure 3 The ROC curve for predicting the occurrence of movement complications based on plasma NfL concentration. As Figure 3 can be seen, when the plasma NfL concentration is greater than 13.66 pg / mL, the probability of patients without movement fluctuations converting to those with movement fluctuations is 0.63, the sensitivity is 88.89%, and the specificity is 35.64%. When the plasma NfL concentration is greater than 13.74 pg / mL, the probability of patients without dyskinesia converting to those with dyskinesia is 0.72, the sensitivity is 51.61%, and the specificity is 87.32%. Therefore, when using plasma NfL levels to predict movement complications (including movement fluctuations and dyskinesia), it has sensitivity and specificity.

[0058] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Any simple modifications and equivalent changes made to the above embodiments based on the technical essence of the present invention all fall within the protection scope of the present invention.

Claims

1. A biomarker for predicting or diagnosing motor complications of Parkinson's disease, characterized in that: By detecting the level of NfL in the blood samples of Parkinson's disease patients, predicting or diagnosing whether Parkinson's disease patients have motor complications.

2. Use of a substance for detecting NfL level in the preparation of a product for predicting or diagnosing the conversion of Parkinson's disease to apathy, characterized in that: For the blood samples of Parkinson's disease patients, check the level of NfL.

3. The application according to claim 2, characterized in that: The blood sample is plasma.

4. The application according to claim 2, characterized in that: The detection method includes single molecule array Simoa detection technology.

5. The application according to claim 2, wherein: The substances include kits, detection reagents, test strips or other substances that can be achieved medically.

6. The application according to claim 2, wherein: When a Parkinson's disease patient initially has no motor fluctuations, when the detected NfL level is greater than 13.66 pg / mL, it is predicted or diagnosed that the probability of this Parkinson's disease patient transforming from initially having no motor fluctuations to having motor fluctuations is relatively high; when a Parkinson's disease patient initially has no dyskinesia, when the detected NfL level is greater than 13.74 pg / mL, it is predicted or diagnosed that the probability of this Parkinson's disease patient transforming from initially having no dyskinesia to having dyskinesia is relatively high.

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