Method for differential diagnosis of parkinson's disease not associated with mutations in GBA1 gene and multiple system atrophy

Abstract

FIELD: neurology; laboratory diagnostics.SUBSTANCE: used for the differential diagnosis of Parkinson's disease not associated with mutations in the GBA1 gene, and multiple system atrophy. The patient's peripheral venous blood is collected, from which mononuclear cells are isolated by gradient centrifugation, followed by their differentiation into a primary culture of macrophages in the presence of the macrophage colony-stimulating growth factor M-CSF to ensure proliferation and differentiation of monocytes into mature macrophages. The obtained blood macrophage cells are applied to 903 filter cards at a concentration of 2×106 cells / ml. Whatman 903 Sample Collection Cards can be used as 903 filter cards. The activity of lysosomal enzymes is determined: glucocerebrosidase GCase, alpha-galactosidase GLA, acid sphingomyelinase ASMase, galactosylceramidase GALC by high-performance liquid chromatography in combination with tandem mass spectrometry. The value of the canonical linear discriminant function CLDF is calculated using the stated formula. If the value of CLDF is ≥ 43.44, the patient is diagnosed with multiple system atrophy. If the value of CLDF is < 43.44, Parkinson's disease is diagnosed in patients who do not have a mutation in the GBA1 gene.EFFECT: method enables reliable and accurate differential diagnosis of Parkinson's disease and multiple system atrophy by assessing the activity of lysosomal enzymes.2 cl, 2 dwg, 2 ex

Description

[0001] The invention relates to the field of medicine, namely to neurology and laboratory diagnostics, and can be used for the differential diagnosis of Parkinson's disease and multiple system atrophy, which are characterized by similar clinical manifestations in the early stages of the disease and belong to the same group of neurodegenerative pathologies - synucleinopathies.

[0002] Parkinson's disease and multiple system atrophy (MSA) are conditions with similar clinical symptoms, making it difficult to differentiate them in the early stages. Developing approaches for the differential diagnosis of Parkinson's disease (PD) and multiple system atrophy (MSA) is a pressing issue in modern neurology.

[0003] The proposed method aims to address the differential diagnosis of sporadic forms of Parkinson's disease not associated with GBA1 gene mutations and multiple system atrophy. Despite their common manifestations, the pathogenesis and prognosis of these diseases differ, requiring accurate laboratory differential diagnosis and the selection of appropriate therapeutic strategies.

[0004] To achieve this goal, an analysis of known solutions aimed at diagnosing Parkinson's disease was carried out, but they do not provide for its differentiation from multiple system atrophy.

[0005] A method for diagnosing Parkinson's disease associated with mutations in the glucocerebrosidase (GBA1) gene is known (RU Patent No. 2750357) [1], based on measuring the concentration of the lysosphingolipid hexosylsphingosine (HexSph) in a primary culture of macrophages obtained from human blood monocytes applied to filter cards at a concentration of 2×10 6cells / ml. Parkinson's disease is diagnosed in carriers of one mutation in the GBA1 gene (heterozygous state) with HexSph concentrations above 32.15 ng / ml. HexSph is a mixture of the lysosphingolipids glucosylsphingosine (GlcSph) and galactosylsphingosine (GalSph). However, this method is designed exclusively for carriers of GBA1 mutations and is not applicable for diagnosing MSA in individuals without such mutations. It also does not differentiate between PD without mutations and multiple system atrophy.

[0006] Also known are approaches based on determining the levels of oligomeric alpha-synuclein in blood plasma (RU Patent No. 2657763) [2], phosphorylated α-synuclein in cerebrospinal fluid, metabolites of the dopamine pathway (L-DOPA and DOUPA) WO2011152699 [3,4], as well as on the analysis of the level of microRNA in human blood cells and biological fluids [5-8].

[0007] However, these methods do not allow for sufficiently reliable differentiation between Parkinson's disease and multiple system atrophy, especially in the early stages of the disease, which necessitates the development of a new diagnostic approach that ensures high specificity and sensitivity.

[0008] At present, the study “Identification of microRNAs for the early diagnosis of Parkinson's disease and multiple system atrophy” is known, which presents a method for the differential diagnosis of Parkinson's disease (PD) and multiple system atrophy (MSA) based on the analysis of the level of microRNAs (miR-16-5p, miR-24-3p, miR-7641, miR-191, miR-671-5p and miR-19b-3p) in the blood cells of patients with these diseases [9], considered as a prototype. The mentioned work showed that the expression profile of these microRNAs differs in patients with PD and MSA, and the microRNAs themselves are involved in the regulation of autophagy processes, protein degradation and control of alpha-synuclein levels, which plays a key role in the pathogenesis of synucleinopathies. The prototype diagnostic method includes the following steps: isolation of microRNA from blood cells, quantitative determination of their level using quantitative real-time PCR, and subsequent interpretation of the obtained data in accordance with threshold values.

[0009] However, the prototype method has a number of limitations that reduce its diagnostic reliability and reproducibility for the following reasons described below.

[0010] Influence of non-specific factors: Plasma microRNA levels are subject to significant fluctuations due to inflammation, stress, diet, and medication, which makes results difficult to interpret and reduces diagnostic accuracy.

[0011] Low stability: Standard microRNA detection methods are not reproducible enough in routine diagnostics, including due to the limited stability of microRNA in biological fluids, which may lead to false results.

[0012] Indirect assessment of pathological processes: microRNAs indirectly reflect disturbances in autophagy and protein degradation, without allowing direct assessment of the degree of lysosomal dysfunction, one of the key mechanisms in the pathogenesis of synucleinopathies.

[0013] Thus, the prototype method for differential diagnosis of PD and MSA using microRNA has limitations that reduce its reliability and accuracy.

[0014] The technical result of the claimed invention is the development of a reliable method for the differential diagnosis of Parkinson's disease and multiple system atrophy by assessing the activity of lysosomal enzymes.

[0015] The technical result of the claimed invention is achieved by creating a method for the differential diagnosis of Parkinson's disease, not associated with mutations in the GBA1 gene, and multiple system atrophy, which consists in the fact that peripheral venous blood is collected from the patient, from which mononuclear cells are isolated by gradient centrifugation, followed by their differentiation into a primary culture of macrophages in the presence of the colony-stimulating factor of macrophage growth M-CSF to ensure proliferation and differentiation of monocytes into mature macrophages, then the obtained blood macrophage cells are applied to 903 filter cards at a concentration of 2 × 10 6cells / ml, and determine the activity of lysosomal enzymes: glucocerebrosidase GCase, alpha galactosidase GLA, acid sphingomyelinase ASMase, galactosylceramidase GALC by high-performance liquid chromatography in combination with tandem mass spectrometry, calculate the value of the canonical linear discriminant function KLDF using the formula:

[0016] KLDF = 44.532 − 0.580 × GALC − 0.203 × ASMase − 0.172 × GCase + 0.226 × GLA,

[0017] where GALC is the activity of the lysosomal enzyme galactosylceramidase of the studied cells of the primary culture of peripheral blood macrophages, mmol / l / h;

[0018] ASMase − activity of the lysosomal enzyme acid sphingomyelinase of the studied cells of the primary culture of peripheral blood macrophages, mmol / l / h;

[0019] GCase − activity of the lysosomal enzyme glucocerebrosidase of the studied cells of the primary culture of peripheral blood macrophages, mmol / l / h;

[0020] GLA − activity of the lysosomal enzyme alpha-galactosidase A of the studied cells of the primary culture of peripheral blood macrophages, mmol / l / h;

[0021] and if the KLDF value is ≥ 43.44, the patient is diagnosed with multiple system atrophy, and if the KLDF value is < 43.44, Parkinson's disease is diagnosed in patients who do not have a mutation in the GBA1 gene.

[0022] It is advisable to use Whatman 903 Sample Collection Cards as 903 filter cards.

[0023] The authors of the claimed invention have experimentally established for the first time that the activity of lysosomal enzymes in the primary culture of peripheral blood macrophages is a sensitive and specific indicator for the differential diagnosis of Parkinson's disease and multiple system atrophy.

[0024] The claimed set of features is based on a comprehensive assessment of the activity of lysosomal enzymes in venous blood, in contrast to the prototype (based on the detection of micro RNA).

[0025] Unlike Russian patent No. 2750357 [1], in which the assessment of the activity of the lysosomal enzyme glucocerebrosidase (GCase) and the concentration of its substrate hexosylsphingosine (HexSph) in the primary culture of peripheral blood macrophages is used to diagnose Parkinson's disease in carriers of mutations in the GBA1 gene (a limited population of patients), the claimed invention is aimed at the differential diagnosis of two different diseases from the group of synucleinopathies - Parkinson's disease and multiple system atrophy - in individuals who do not have mutations in the GBA1 gene.

[0026] Currently, lysosomal enzymes such as glucocerebrosidase (GCase), alpha-galactosidase (GLA), acid sphingomyelinase (ASMase), and galactosylceramidase (GALC) have been widely used for the diagnosis of lysosomal storage diseases (Gaucher disease, Fabry disease, Niemann-Pick type A / B, Krabbe disease, respectively) using multiplex panels of lysosomal enzyme activity and substrate profiling by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS / MS) [10-14].

[0027] Description of the method

[0028] The method was developed based on the study to evaluate the activity of lysosomal enzymes (glucocerebrosidase (GCase), alpha-galactosidase (GLA), acid sphingomyelinase (ASMase), and galactosylceramidase (GALC)) in the primary culture of macrophages in three groups of patients: with multiple system atrophy (N=10, mean age: 61.4±9.96 years, mean age of onset 58.0±8.55, 5 men, 5 women); with sporadic Parkinson's disease (N=22, mean age: 65.82±10.04 years, mean age of onset 54.90±10.49, 11 men, 11 women); age-matched neurologically healthy individuals (N=43, mean age: 64.85±8.97, 24 men, 19 women) - control group. The activity of lysosomal enzymes (glucocerebrosidase (GCase), acid sphingomyelinase (ASMase), galactosylceramidase (GALC)) is assessed.

[0029] When analyzing the GCase activity in the primary culture of peripheral blood macrophages (median (min-max), mmol / l / h) in the group of patients with multiple system atrophy (2.89 (0.35-19.13) mmol / l / h), a statistically significant decrease in GCase activity was found compared with the group of patients with Parkinson's disease (8.02 (2.04-54.24) mmol / l / h) (p=0.00086) and the control group (6.72 (0.07-48.24) mmol / l / h) (p=0.013).

[0030] When analyzing ASMase activity in primary culture of peripheral blood macrophages (median (min-max), mmol / l / h) in the group of patients with multiple system atrophy (0.68 (0.01-4.27) mmol / l / h), a statistically significant decrease in ASMase activity was revealed compared with the group of patients with Parkinson's disease (2.05 (0.25-35.9) mmol / l / h) (p=0.022) and the control group (4.87 (0.09-55.87) mmol / l / h) (p<0.0001).

[0031] When analyzing GALC activity in the primary culture of peripheral blood macrophages (median (min-max), mmol / l / h) in the group of patients with multiple system atrophy (1.14 (0.18-3.67) mmol / l / h), a statistically significant decrease in GALC activity was revealed compared with the group of patients with Parkinson's disease (2.42 (0.8-12.74) mmol / l / h) (p=0.0076) and the control group (2.44 (0.11-15.96) mmol / l / h) (p=0.0068).

[0032] When analyzing the GLA activity in the primary culture of peripheral blood macrophages (median (min-max), mmol / l / h) in the group of patients with multiple system atrophy (9.24 (0.86-15.44) mmol / l / h), no changes in GLA activity were found compared with the group of patients with Parkinson's disease (4.00 (1.44-70.14) mmol / l / h) (p> 0.05) and the control group (2.44 (0.11-15.96) mmol / l / h) (p> 0.05).

[0033] To assess the possibility of using the activity of lysosomal enzymes - glucocerebrosidase (GCase), alpha-galactosidase (GLA), acid sphingomyelinase (ASMase) and galactosylceramidase (GALC) - as a comprehensive approach for the differential diagnosis of Parkinson's disease and multiple system atrophy, a method of quantitative analysis of their activity in the primary culture of human peripheral blood macrophages obtained from monocytes and precipitated on filter cards at a concentration of 2×10 6 cells / ml.

[0034] To evaluate the diagnostic values ​​of lysosomal enzyme activities in the differential diagnosis of Parkinson's disease (PD) and multiple system atrophy (MSA), canonical linear discriminant analysis (LDA) with leave-one-out cross-validation (LOOCV) was performed. The model was trained using predictors including the activities of glucocerebrosidase (GCase), alpha-galactosidase (GLA), acid sphingomyelinase (ASMase), and galactosylceramidase (GALC). The diagnostic accuracy was assessed using leave-one-out cross-validation (LOOCV). The trained classification model achieved an area under the curve (AUC) of AUC = 0.84 (95% CI: 0.70-0.95) (bootstrap estimation, 1000 replicates). The difference between the model and random classification was statistically significant (p = 0.00066).

[0035] Based on the trained model, the equation of the canonical linear discriminant function (CLDF) was obtained:

[0036] KLDF = 44.532-0.580×GALC-0.203×ASMase-0.172×GCase+0.226×GLA,

[0037] where:

[0038] GALC - galactosylceramidase activity, in mM / l / h;

[0039] ASMase - acid sphingomyelinase activity, in mmol / l / h;

[0040] GCase - glucocerebrosidase activity, in mmol / l / h;

[0041] GLA - alpha-galactosidase A activity, in mmol / l / h;

[0042] 44,532 - constant;

[0043] 0.580, 0.203, 0.172, 0.226 - coefficients for the corresponding activities of lysosomal enzymes.

[0044] The KLDF value is calculated for each patient. A patient is classified as having multiple system atrophy if the KLDF value is equal to or greater than 43.44, and as having Parkinson's disease if the value is less than 43.44.

[0045] Thus, the results of the analysis demonstrate the feasibility of reliably classifying patients with Parkinson's disease and multiple system atrophy based on the lysosomal enzyme activity profile of peripheral blood macrophages. This approach differs from traditional diagnostic methods based on visual assessment and clinical scoring and can be used as an auxiliary tool in clinical practice, particularly in the early stages of the disease.

[0046] Fig. 1 shows a comparative assessment of the activity of lysosomal enzymes in the primary culture of peripheral blood macrophages in patients with Parkinson's disease (PD), multiple system atrophy (MSA) and in neurologically healthy individuals of the control group.

[0047] The activities of the enzymes glucocerebrosidase (GCase), alpha-galactosidase (GLA), acid sphingomyelinase (ASMase), and galactosylceramidase (GALC) are displayed as diagrams with median values ​​and ranges of variation (min-max). The graphs demonstrate a statistically significant decrease in the activities of GCase, ASMase, and GALC in the MSA group compared to PD patients and the control group, as confirmed by the p-values. No statistically significant differences were found for GLA between the groups. The diagrams clearly illustrate the differences in enzyme activities in the studied cohorts.

[0048] Figure 2 shows the evaluation of the diagnostic value of lysosomal enzyme activity using receiver operating characteristic (ROC) analysis for the differential diagnosis of Parkinson's disease and multiple system atrophy. The ROC curve was plotted using canonical linear discriminant analysis (LDA) with leave-one-out cross-validation (LOOCV). The area under the curve (AUC) was 0.84 (95% confidence interval: 0.70-0.95). The sensitivity of this method was 94%, and the specificity was 70%. The graph also includes a random classification line for visual comparison, demonstrating the advantage of the lysosomal enzyme activity-based model.

[0049] The method is carried out, for example, as follows.

[0050] Blood collection for each patient is performed from a vein into three 9 ml EDTA tubes. The mononuclear fraction is obtained from 24 ml by gradient centrifugation in a Ficoll solution (p=1.077, Ficoll-Paque PLUS, GE Healthcare UK Limited, UK) at 1600 rpm according to the method described in

[15] , followed by washing the resulting cell suspension in a sodium phosphate buffer solution (PBS) and centrifugation at 3000 rpm for 10 minutes. The resulting mononuclear cells are resuspended in culture medium (RPMI Medium, Gibco, USA) supplemented with 10% bovine serum (BioloT, Russia) and 1% penicillin streptomycin (Penicillin Streptomycin, Gibco, USA) and transferred to Petri dishes (5 ml per dish). The cells are cultured in a 5% CO2 incubator at 37°C. After 2 hours of culture, non-adherent cells are removed from the Petri dishes by rinsing with PBS.Then, monocytes are cultured in a culture medium supplemented with M-CSF (Macrophage Colony-Stimulating Factor, Gibco, USA) at a final concentration of 10 ng / ml for 5 days with daily medium replacement according to the protocol described by the authors in

[16] . After culturing, the cells are washed twice in PBS and centrifuged at 1500 rpm for 5 minutes. Maturation of peripheral blood macrophages is confirmed by light microscopy, as well as flow cytometry using specific CD14+ and CD68+ antibodies. The suspension of the obtained macrophages is applied to special circles on filter cards (Whatman 903 Sample Collection Cards, Germany) at a concentration of 2×10. 6cells / ml. Next, the filter cards with the test samples applied to them are dried for 2 hours at room temperature and stored at + 4°C until further use for no more than one month. The activity of lysosomal enzymes (glucocerebrosidase (GCase), acid sphingomyelinase (ASMase), galactosylceramidase (GALC), alpha-galactosidase (GLA)) is assessed by high-performance liquid chromatography and tandem mass spectrometry (HPLC-MS / MS) according to the protocol described previously

[14] with modifications, in particular, measuring in primary macrophage culture cells at a concentration that is applied to filter cards at a concentration of 2×10 6 cells / ml.

[0051] Clinical example 1. A 68-year-old female patient was diagnosed with PD according to the 2015 MDS criteria

[17] . Enzyme activities were determined and their values ​​were substituted into the formula:

[0052] KLDF=44.532-0.580×3.82-0.203×4.88-0.172×13.25+0.226×8.14.

[0053] According to the criterion established in the method, a KLDF value of less than 43.44 predicts PD. The obtained value (40.89) is below the diagnostic threshold, which allowed us to confirm PD in the patient with a high degree of certainty.

[0054] Clinical example 2. A 70-year-old female patient was diagnosed with MSA according to the 2022 criteria

[18] . Enzyme activities were determined and the values ​​were substituted into the formula:

[0055] KLDF=44.532-0.580×0.48-0.203×2.11-0.172×6.25+0.226×11.28.

[0056] According to the criterion established in the method, a KLDF value ≥ 43.44 predicts MSA. The obtained value (45.30) is above the diagnostic threshold, which allowed us to confirm MSA in the patient with a high degree of certainty.

[0057] Conclusion: The proposed method can serve as a method for differential diagnosis of Parkinson's disease and multiple system atrophy in the early stages.

[0058] Bibliography

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Claims

1. A method for the differential diagnosis of Parkinson's disease not associated with mutations in the GBA1 gene and multiple system atrophy, which consists of collecting peripheral venous blood from a patient, from which mononuclear cells are isolated by gradient centrifugation, followed by their differentiation into a primary culture of macrophages in the presence of the colony-stimulating factor of macrophage growth M-CSF to ensure proliferation and differentiation of monocytes into mature macrophages, then the obtained blood macrophage cells are applied to 903 filter cards at a concentration of 2×10 6 cells / ml and determine the activity of lysosomal enzymes: glucocerebrosidase GCase, alpha-galactosidase GLA, acid sphingomyelinase ASMase, galactosylceramidase GALC by high-performance liquid chromatography in combination with tandem mass spectrometry, calculate the value of the canonical linear discriminant function KLDF using the formula: KLDF=44.532−0.580×GALC−0.203×ASMase−0.172×GCase+0.226×GLA, where GALC is the activity of the lysosomal enzyme galactosylceramidase of the studied cells of the primary culture of peripheral blood macrophages, mmol / l / h; ASMase is the activity of the lysosomal enzyme acid sphingomyelinase of the studied cells of the primary culture of peripheral blood macrophages, mmol / l / h; GCase is the activity of the lysosomal enzyme glucocerebrosidase of the studied cells of the primary culture of peripheral blood macrophages, mmol / l / h; GLA is the activity of the lysosomal enzyme alpha-galactosidase A of the studied cells of the primary culture of peripheral blood macrophages, mmol / l / h; and if the KLDF value is ≥43.44, the patient is diagnosed with multiple system atrophy, and if the KLDF value is <43.44, Parkinson's disease is diagnosed in patients who do not have a mutation in the GBA1 gene.

2. The method according to claim 1, wherein the filter cards 903 are Whatman 903 Sample Collection Cards.

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