VEGFA Gene Expression Levels in Patients with Parkinson’s Disease and First-Degree Relatives

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Augste, K. Smešný Trtková, K. Feichtingerová, A. Kochanová, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8433576/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Background: Vascular endothelial growth factor A (VEGFA) contributes to angiogenesis and exerts neurotrophic and neuroprotective effects. Altered VEGFA expression has been implicated in neurodegenerative processes, including Parkinson´s disease (PD), yet findings from peripheral transcriptomic studies remain inconsistent. Data on individuals at increased risk, particularly first-degree relatives (FDRs), are limited. Objective: To evaluate peripheral blood VEGFA mRNA expression in patients with PD and their asymptomatic FDRs compared with healthy controls, and to assess age-related patterns and diagnostic performance. Methods: Peripheral blood VEGFA expression was analyzed in 123 participants (PD patients, FDRs, healthy controls) using one-step RT-qPCR normalized to B2M . Relative expression values were log-transformed (logVEGFA). Group differences, age correlations, and diagnostic discrimination were assessed using ANOVA, Pearson correlation, and receiver operating characteristic (ROC) analysis. Results: Both PD patients and FDRs showed significantly higher logVEGFA levels than healthy controls (p = 0.004), with the most pronounced differences observed in participants aged ≤ 60 years (p < 0.001). A positive correlation between age and logVEGFA was detected only in healthy controls. In participants aged ≤ 60 years, logVEGFA demonstrated excellent discrimination between PD patients and healthy individuals (AUC 0.897; 95% CI 0.771–1.000; sensitivity 100%, specificity 73.3%). Conclusions: Peripheral VEGFA expression is elevated in both PD patients and FDRs, particularly in younger individuals. Age-stratified VEGFA assessment may support early identification of individuals at risk of PD and complement existing prodromal biomarkers, pending longitudinal validation. Health sciences/Biomarkers Health sciences/Diseases Health sciences/Neurology Biological sciences/Neuroscience Parkinson’s disease VEGFA gene expression biomarkers first-degree relatives RT-qPCR Figures Figure 1 Figure 2 Figure 3 Introduction Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and also exerts important neurotrophic and neuroprotective functions within the central nervous system. Among the VEGF isoforms, VEGF-A is the most extensively studied and has been linked to neuronal survival, inhibition of apoptosis, and neural regeneration, making it a molecule of interest in neurodegenerative disorders such as Parkinson’s disease (PD) (3–5). PD is a progressive neurodegenerative condition characterized by degeneration of dopaminergic neurons in the substantia nigra pars compacta. Its multifactorial pathogenesis involves mitochondrial dysfunction, oxidative stress, neuroinflammation, and impairment of endogenous neuroprotective mechanisms (6,7). Several transcriptomic studies have examined peripheral VEGFA expression in PD, though their findings remain inconsistent (8–10). Protein-level studies also report heterogeneous results: VEGF-A is often elevated in post-mortem brain tissue or cerebrospinal fluid, whereas peripheral protein concentrations are frequently unchanged (11–13). These discrepancies may reflect methodological variability, central–peripheral compartment differences, or weak mRNA–protein correlation driven by post-transcriptional and translational regulation (14,15). Demographic factors, particularly age, may further contribute to variability in measured VEGFA levels (16). Despite growing interest in molecular biomarkers of preclinical neurodegeneration, little is known about peripheral VEGFA expression in individuals at elevated risk of PD, particularly first-degree relatives (FDRs) (17,18). FDRs represent a key population for biomarker studies not only because of their higher lifetime risk, but also because subtle motor and non-motor abnormalities - including hyposmia, anxiety, or REM sleep behavior disorder - have been reported even in asymptomatic relatives. Such early alterations suggest underlying biological changes that precede clinical onset (19–22). Experimental data further support a potential role of VEGF signaling in early neurodegenerative processes (5). In this study, we investigated peripheral blood VEGFA expression in patients with PD, their asymptomatic first-degree relatives, and healthy controls. We also examined age-related expression patterns to determine whether VEGFA may serve as an early biomarker of neurodegeneration in at-risk individuals. Materials and Methods Participants and study design Patients with a clinically confirmed diagnosis of Parkinson’s disease (PD) were recruited according to the Movement Disorder Society (MDS) diagnostic criteria (23). Disease severity was evaluated using the Movement Disorder Society–sponsored revision of the Unified Parkinson’s Disease Rating Scale (MDS-UPDRS) and the modified Hoehn and Yahr scale (24). Asymptomatic first-degree relatives (FDRs), defined as siblings or children of PD patients, with no history of neurological disease were included. Healthy controls had no personal or family history of PD or other neurological disorders. Inclusion and exclusion criteria for all study groups are listed in Table 1. Sample collection and processing Peripheral venous blood samples were collected into K₃EDTA tubes (SARSTEDT AG & Co. KG, Nümbrecht, Germany) and immediately stored at −80°C until processing to minimize RNA degradation. After thawing, total RNA was isolated from 200 µL of frozen whole blood using the NucleoSpin® RNA Blood Kit (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany) according to the manufacturer’s instructions. RNA purity and quantity were determined spectrophotometrically using a NanoDrop 2000 instrument (Thermo Fisher Scientific, Waltham, MA, USA). Samples with an A260/280 ratio outside the 1.8–2.0 range were excluded. RT-qPCR analysis Quantification of VEGFA expression was performed using reverse transcription quantitative polymerase chain reaction (RT-qPCR) with the KAPA PROBE FAST Universal One-Step qRT-PCR Master Mix (2X) Kit (Roche Diagnostics GmbH, Mannheim, Germany). TaqMan probes were used for the target gene VEGFA (Assay ID: Hs03929054_s1) and the reference gene B2M (Assay ID: Hs00984230_m1). Each reaction had a total volume of 20 µL, including 2 µL of isolated RNA. Amplification was performed on a qTOWER³ touch/qTOWER³ G touch Real-Time PCR Thermal Cycler (Analytik Jena GmbH+Co. KG, Jena, Germany) using qPCRsoft 4.1 software. The thermal cycling protocol consisted of: reverse transcription at 42 °C for 5 min, polymerase activation at 95 °C for 3 min, followed by 40 cycles of 95 °C for 3 s and 60 °C for 30 s. Each sample was analyzed in two to four technical replicates. Samples with a Ct standard deviation > 0.25 across replicates were excluded. In this study, ‘ VEGFA expression” denotes peripheral blood mRNA quantified by one-step RT-qPCR and normalized to the reference gene B2M . The results were expressed as the relative expression of VEGFA to B2M , calculated using the 2^-ΔΔCt method (25,26). All relative expression values were natural-log transformed (logVEGFA) for subsequent analysis. Statistical analysis For each participant, the geometric mean of the technical replicates of relative VEGFA expression was calculated (due to proportional data). These values were then natural-log transformed (the variable logVEGFA) to follow normal distribution as confirmed by the Shapiro–Wilk test. Subsequent analyses used logVEGFA as specified below. The baseline characteristics were analyzed using descriptive statistics. Continuous variables are presented as the mean ± standard deviation, categorical variables as absolute and relative frequencies (%). Between-group differences for continuous variables were analyzed with the analysis of variance (ANOVA) with the Games-Howell or Tukey post-hoc test. Differences between groups for categorical variables were assessed using the chi-square test or Fisher’s exact test, as appropriate. The correlation between age and logVEGFA was assessed using Pearson’s correlation coefficient. Receiver operating characteristic (ROC) analysis was performed to determine the optimal cut-off value for VEGFA expression distinguishing PD patients. The cut-off was selected by maximizing Youden’s index. All statistical analyses were performed using IBM SPSS 30.0 statistical software. Two-tailed p-values < 0.05 were considered statistically significant. Ethics Participants were recruited between January 2023 and December 2024 at the Faculty of Medicine, University of Ostrava, and collaborating neurology outpatient clinics. The study was approved by the Ethics Committee of the Faculty of Medicine, University of Ostrava (Approval No. SGS02/LF/2023) and conducted in accordance with the Declaration of Helsinki (2013 revision). Written informed consent was obtained from all participants. Data processing complied with the EU General Data Protection Regulation (GDPR, 2016/679). Results A total of 123 subjects (64 males, mean age: 57.5 ± 14.6 years) were enrolled in the study: 41 patients with clinically established PD (29 males; mean age: 66.3 ± 9.6 years), 52 asymptomatic FDRs (22 males; mean age: 50.2 ± 13.9 years), and 30 healthy controls (13 males, mean age: 58.1 ± 14.8 years). All details pertaining to the studied sample are available in Table 2. Given the observed correlation with age and statistically significant differences between the groups, the cohort was subsequently stratified into two age categories: 60 years. Comparison of logVEGFA values across all groups revealed significant differences (p = 0.004, ANOVA Welch test), with the lowest expression observed in the group of healthy controls (Table 2). Post hoc testing using the Games-Howell test showed significant differences between healthy controls and patients with PD (p = 0.003), as well as between healthy controls and FDRs (p = 0.011). No significant differences were observed between patients with PD and their relatives (p = 0.773; Table 3, Figure 1a). When analyzing age-stratified subgroups, differences in logVEGFA expression between groups persisted only in younger participants (p < 0.001), whereas they were no longer statistically significant among older individuals (p = 0.643; Tables 2 and 3, Figure 1b). The healthy control group exhibited a moderate linear trend of increasing logVEGFA with age (Pearson’s correlation coefficient r = 0.571, p < 0.001), whereas no such significant correlation was observed in patients with PD or FDRs (Figure 2). Based on the previous results, ROC analysis was conducted to assess the diagnostic performance of logVEGFA (i.e., relative VEGFA expression) in distinguishing patients with PD from healthy controls. In participants aged ≤ 60 years, where the differences between groups were most pronounced, VEGFA expression (logVEGFA) showed an excellent ability to distinguish patients with PD from healthy individuals. The area under the ROC curve (AUROC) reached 0.897 (95% confidence interval: 0.771–1.000, p < 0.001). According to the maximal Youden’s index, the optimal cut-off value was determined to be logVEGFA = –1.346 (i.e., 0.260 for relative expression VEGFA ), with 100% sensitivity, 73.3 % specificity, and an overall accuracy of 84.6% (Figure 3). Discussion In this study, we demonstrated that peripheral VEGFA expression is elevated not only in patients with PD but also in asymptomatic FDRs compared with healthy controls (Tables 2 and 3). Age-stratified analysis revealed that these group differences in VEGFA expression were most pronounced in individuals aged ≤ 60 years. In healthy controls, age-related decline is well documented for several non-motor domains relevant to prodromal PD, including olfaction and sleep (27–30). In contrast, olfactory abnormalities tend to persist across age strata in PD and at-risk cohorts (31,32). We observed an age-associated increase in peripheral VEGFA expression exclusively among healthy controls, a trend consistent with age-linked low-grade inflammation and leukocyte compositional changes rather than endothelial expression shifts (33). The absence of this association in PD patients and FDRs suggests that early neurodegenerative processes may interfere with physiological, age-associated compensatory mechanisms. These findings underscore the importance of incorporating age stratification when evaluating molecular biomarkers in prodromal or early PD (32,34). Prior studies assessing circulating VEGF-A protein levels have shown heterogeneous results, with several reporting no significant differences between PD patients and controls (13). Such variability may reflect heterogeneity in cohorts, methodological differences, or the inherently limited correlation between mRNA expression and protein abundances due to post-transcriptional and translational regulation. Our study specifically focused on VEGFA transcript levels in a relatively younger population (≤ 60 years), which may yield better sensitivity for detecting early disease-related molecular changes. The dual role of VEGF-A in the central nervous system adds biological plausibility to these findings. VEGF-A supports angiogenesis, enhances regional cerebral blood flow, and promotes dopaminergic neuron survival, all of which have demonstrated protective effects in experimental PD models (5). Conversely, dysregulated VEGF-A signaling has been linked to blood-brain barrier dysfunction and neuroinflammatory processes implicated in PD pathogenesis (3,12). Whether the elevated VEGFA expression observed in FDRs represents an adaptive compensatory response or an early molecular hallmark of neurodegeneration remains unclear. FDRs constitute a biologically relevant at-risk population due to their increased lifetime risk and their tendency to exhibit subtle motor and non-motor abnormalities - such as hyposmia, anxiety, depression, or REM sleep behavior disorder - even in the absence of clinically manifest PD (19–22). Imaging studies further support early biological alterations, with dopaminergic deficits on DAT-SPECT reported in hyposmic relatives of PD patients (22,34). Family history also modifies disease risk, as siblings of patients with early-onset PD are more likely to develop PD and may present at a younger age (35). Elevated peripheral VEGFA expression in FDRs, as observed in our study, raises the question of whether this pattern reflects an adaptive compensatory response or an early molecular component of the pathogenic process. A common genetic variant in VEGFA (rs3025039) has been identified as a risk factor for sporadic PD (5), suggesting that genetic susceptibility and regulatory dynamics of VEGFA expression may influence vulnerability to neurodegeneration. From a biomarker perspective, our findings suggest that peripheral VEGFA expression, particularly when integrated with established prodromal markers such as hyposmia or REM sleep behavior disorder and with probability-based risk algorithms, may contribute to improved risk stratification. Previous studies have reported increased VEGF-A or VEGFR-1 levels in dopaminergic neurons and astrocytes of the substantia nigra (5,11), as well as elevated VEGF in cerebrospinal fluid (12). Our results extend these observations by demonstrating that asymptomatic FDRs also show elevated peripheral VEGFA levels, potentially reflecting either early compensatory activation of VEGF signaling or an early step in disease evolution. Our findings also suggest that peripheral VEGFA expression has potential utility as an early biomarker of PD risk. In participants aged ≤ 60 years , we identified a VEGFA expression threshold (logVEGFA = –1.346) that distinguished patients with PD from healthy controls with high accuracy (sensitivity, 100%; specificity, ~73.3%). Because peripheral expression can be influenced by systemic variables, this biomarker is likely to be most informative when combined with additional prodromal markers or biological measures. Future longitudinal studies are needed to determine whether elevated VEGFA expression in at-risk individuals predicts conversion to PD and whether interventions - such as exercise or neuroprotective strategies - may modify VEGFA expression and alter disease trajectory. Study limitations This study has several limitations that should be considered when interpreting the results. First, the age distribution of the cohort may have influenced the findings, as early-onset PD (≤ 50 years) is relatively rare, and only a small proportion of patients with PD are diagnosed before the age of 60 (36,37). This may amplify age-related effects in subgroup analyses. Second, the logVEGFA values reflect relative gene expression normalized to B2M rather than circulating VEGF-A protein levels; given that mRNA–protein correlations are typically low and gene-specific, transcript levels may not directly reflect systemic protein concentrations (14). As an additional methodological note, RT-qPCR data were normalized to a single reference gene, B2M . Although B2M showed stable expression in our dataset, the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines recommend using at least two validated reference genes to minimize normalization bias. Finally, the genetic analysis was not comprehensive because no systematic screening was performed for known PD-related mutations. Although monogenic forms represent only approximately 5–10% of all PD cases and occur more frequently in early-onset forms, testing a broader panel of genes, such as LRRK2 , PRKN , PINK1 , or DJ1 , could have provided further insight into the relationship between genetic predisposition and VEGFA expression (38–40). Despite these limitations, the principal finding remains: both PD patients and their first-degree relatives exhibit elevated peripheral VEGFA expression compared with healthy controls. Taken together with age-stratified analyses and ROC results, these findings provide preliminary support for VEGFA as a candidate biomarker for early PD risk. Conclusion Peripheral VEGFA expression was significantly higher in both patients with PD and their first-degree relatives compared with healthy controls, with the clearest group differences observed in individuals aged ≤ 60 years. Only healthy controls showed an age-related increase in VEGFA expression, suggesting disruption of physiological regulation in PD and at-risk individuals. In younger participants, VEGFA expression demonstrated excellent discriminatory ability, supporting its potential utility as an early biomarker when combined with other prodromal measures. Longitudinal studies are needed to determine its predictive value for conversion to PD. Declarations Acknowledgement We thank all participants for their involvement in this study. Funding This work was supported by the National Institute for Neurological Research, Programme EXCELES (Project No. LX22NPO5107), funded by the European Union – Next Generation EU, and by the University of Ostrava (Grant SGS02/LFOU/2023). The funders had no role in study design, data collection, data analysis, data interpretation, or manuscript preparation. Competig Interests. The authors declare no competing interests. Author Contributions. EA, DSk and PB conceived and designed the study. EA, FS and TM collected clinical data and samples. KTS, KF and AK performed laboratory analyses. EA and DSa conducted statistical analyses. EA, ZH, PD and DSa drafted the manuscript. All authors contributed to data interpretation, critically revised the manuscript, and approved the final version. Data Availability Statement. The data supporting the findings of this study are available from the corresponding author upon reasonable request. References Calvo PM, Hernández RG, de la Cruz RR, et al. 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Inclusion and exclusion criteria for subjects enrolled to the prospective study Study group Inclusion criteria Exclusion criteria Patients with Parkinson´s disease Clinically established PD (Postuma RB et al 2015) Diagnosed neurological or psychiatric disease except PD; known genetic mutation associated with PD; oncological treatment, ongoing infection or current tretment with immunosuppressants or corticosteroids Male or female Known impairment of olfactory function (other than PD etiology) Age ≥ 20 years PD First-degree relative Male or female Diagnosed neurological or psychiatric disease; known genetic mutation associated with PD; oncological treatment, ongoing infection or current tretment with immunosuppressants or corticosteroids Age ≥ 20 years Known impairment of olfactory functions Healthy subjects Subject without any known chronic disease Diagnosed neurological or psychiatric disease; oncological treatment, ongoing infection or current tretment with immunosuppressants or corticosteroids Male or female Known impairment of olfactory functions Age ≥ 20 years PD – Parkinson´s disease Table 2. Baseline patient characteristics. PD Relatives Controls P value All participants Number of participants; n 41 52 30 NA Age; mean ±SD (years) 66.3 ± 9.6 50.2 ± 13.9 58.1 ± 14.8 < 0.001 a Male sex; n (%) 29 (70.7) 22 (42.3) 13 (43.3) 0.013 b logVEGFA; mean ±SD -0.514 ± 0.590 -0.605 ± 0.687 -1.240 ± 1.024 0.004 c Age ≤ 60 years Number of participants; n 11 41 15 Age; mean ±SD (years) 53.9 ± 6.1 45.8 ± 11.9 45.5 ± 9.6 0.020 a Male sex; n (%) 7 (63.6) 19 (46.3) 6 (40.0) 0.471 b logVEGFA; mean ±SD -0.316 ± 0.534 -0.637 ± 0.703 -1.717 ± 0.991 60 years Number of participants; n 30 11 15 Age; mean ±SD (years) 70.8 ± 6.0 66.6 ± 6.2 70.7 ± 4.7 0.067 a Male sex; n (%) 22 (73.3) 3 (27.3) 7 (46.7) 0.019 b logVEGFA; mean ±SD -0.587 ± 0.601 -0.484 ± 0.642 -0.762 ± 0.838 0.643 c a Kruskal-Wallis test; b Chi-square test; c ANOVA (the Welch test); PD – Parkinson´s disease; SD – standard deviation; VEGFA – vascular endothelial growth factor isoform A Table 3. Pairwise comparisons of logVEGFA between groups (all participants and separately participats ≤ 60 years). All participants Age ≤ 60 years p -value a p -value b Healthy controls vs. PD patients 0.003 < 0.001 Healthy controls vs. PD relatives 0.011 < 0.001 PD patients vs. PD relatives 0.773 0.426 PD – Parkinson’s disease, a adjusted by the Games-Howell post-hoc test, b adjusted by the Tukey post-hoc test Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 16 May, 2026 Reviews received at journal 24 Mar, 2026 Reviewers agreed at journal 17 Mar, 2026 Reviewers invited by journal 16 Mar, 2026 Editor invited by journal 30 Dec, 2025 Editor assigned by journal 26 Dec, 2025 Submission checks completed at journal 26 Dec, 2025 First submitted to journal 23 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8433576","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":608484917,"identity":"257e9c6d-e22c-469f-8eb2-a51ea9aee733","order_by":0,"name":"E. Augste","email":"","orcid":"","institution":"University of Ostrava","correspondingAuthor":false,"prefix":"","firstName":"E.","middleName":"","lastName":"Augste","suffix":""},{"id":608484932,"identity":"a2162d9f-4ccf-42c5-bdda-e8fb01178e75","order_by":1,"name":"K. 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Dušek","email":"","orcid":"","institution":"Charles University","correspondingAuthor":false,"prefix":"","firstName":"P.","middleName":"","lastName":"Dušek","suffix":""},{"id":608484957,"identity":"6ffe2bdb-14dc-4590-9e0d-0dbf2570e195","order_by":11,"name":"D. Šalounová","email":"","orcid":"","institution":"University of Ostrava","correspondingAuthor":false,"prefix":"","firstName":"D.","middleName":"","lastName":"Šalounová","suffix":""},{"id":608484961,"identity":"fc66946f-70fc-40f0-8981-804a6a5bb9dc","order_by":12,"name":"D. Školoudík","email":"","orcid":"","institution":"University of Ostrava","correspondingAuthor":false,"prefix":"","firstName":"D.","middleName":"","lastName":"Školoudík","suffix":""},{"id":608484972,"identity":"f5767660-7f7c-4369-86a1-8d205754cd11","order_by":13,"name":"P. Bártová","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYLACxgYo4wNx6pkhWnhAWmeQrIWZhxgN5uz9xz783GGXb8/eY/jZ5o+NvTn76QSGH39wa7HsOcw8s/dMsmUPzxlj6dy2NGbLntwNjD147DO4kczMwNvGbMAjkZYgndtwmM3gQO4GZgYJPFruP2Zm/NtWD9KS/Nviz38eg/NvgVoM8NnCzMzM23YYqCX5mDQD2wEJgxsgWxLw+SXZmFn2zHEDnjOHj1n2tiUbGNx4u+FgzwHcWszZDz5mfLuj2oC9vbH5xo8/dvYG53M3PsAXYtjdjMcOXFpGwSgYBaNgFCADAIjCTawn211+AAAAAElFTkSuQmCC","orcid":"","institution":"University of Ostrava","correspondingAuthor":true,"prefix":"","firstName":"P.","middleName":"","lastName":"Bártová","suffix":""}],"badges":[],"createdAt":"2025-12-23 12:23:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8433576/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8433576/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105034983,"identity":"bccfc335-629e-46e0-a427-e71d8277e4f6","added_by":"auto","created_at":"2026-03-20 07:25:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":49355,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of logVEGFA expression across study groups.\u003c/p\u003e\n\u003cp\u003e(a) Boxplots showing log-transformed \u003cem\u003eVEGFA\u003c/em\u003eexpression levels in patients with Parkinson’s disease (PD), first-degree relatives (FDRs), and healthy controls (HC), across all participants regardless of age.\u003c/p\u003e\n\u003cp\u003e(b) Boxplots showing logVEGFA expression stratified by age group (≤ 60 years vs. \u0026gt; 60 years) across the three study groups (PD, FDRs, HC). Horizontal lines indicate median values.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8433576/v1/9f5a13f8257a478621364781.png"},{"id":104996120,"identity":"5b2daed2-00d6-46be-9e91-f645cf89b21c","added_by":"auto","created_at":"2026-03-19 16:11:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":86648,"visible":true,"origin":"","legend":"\u003cp\u003eAssociation between age and logVEGFA expression.\u003c/p\u003e\n\u003cp\u003eScatterplots with fitted linear regression lines illustrating the relationship between age and logVEGFA in patients with PD, first-degree relatives (FDRs), and healthy controls (HC).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8433576/v1/cbd74510d32c3066f71311b1.png"},{"id":104996181,"identity":"835a543b-83a8-44b1-aa87-abd2014b10ac","added_by":"auto","created_at":"2026-03-19 16:11:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":43501,"visible":true,"origin":"","legend":"\u003cp\u003eROC curve for logVEGFA in distinguishing PD patients from healthy controls (≤ 60 years).\u003c/p\u003e\n\u003cp\u003eReceiver operating characteristic (ROC) curve showing discrimination performance of logVEGFA in participants aged ≤ 60 years. The area under the curve (AUC) and 95% confidence intervals are reported in the text.\u003c/p\u003e\n\u003cp\u003eAbbreviations: PD – Parkinson’s disease; ROC – receiver operating characteristic.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8433576/v1/589d9b9a134d7ef07e7075c2.png"},{"id":105036712,"identity":"32269688-2723-434f-a9b9-d77915c2c139","added_by":"auto","created_at":"2026-03-20 07:35:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1054742,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8433576/v1/3355d5ab-8d7b-43bb-9bc3-3f40505b4f27.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"VEGFA Gene Expression Levels in Patients with Parkinson’s Disease and First-Degree Relatives","fulltext":[{"header":"Introduction","content":"\u003cp\u003eVascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and also exerts important neurotrophic and neuroprotective functions within the central nervous system. Among the VEGF isoforms, \u003cem\u003eVEGF-A\u003c/em\u003e is the most extensively studied and has been linked to neuronal survival, inhibition of apoptosis, and neural regeneration, making it a molecule of interest in neurodegenerative disorders such as Parkinson\u0026rsquo;s disease (PD)\u003cem\u003e\u0026nbsp;\u003c/em\u003e(3\u0026ndash;5).\u003c/p\u003e\n\u003cp\u003ePD is a progressive neurodegenerative condition characterized by degeneration of dopaminergic neurons in the substantia nigra pars compacta. Its multifactorial pathogenesis involves mitochondrial dysfunction, oxidative stress, neuroinflammation, and impairment of endogenous neuroprotective mechanisms (6,7). Several transcriptomic studies have examined peripheral \u003cem\u003eVEGFA\u003c/em\u003e expression in PD, though their findings remain inconsistent (8\u0026ndash;10).\u0026nbsp;Protein-level studies also report heterogeneous results: \u003cem\u003eVEGF-A\u003c/em\u003e is often elevated in post-mortem brain tissue or cerebrospinal fluid, whereas peripheral protein concentrations are frequently unchanged (11\u0026ndash;13).\u0026nbsp;These discrepancies may reflect methodological variability, central\u0026ndash;peripheral compartment differences, or weak mRNA\u0026ndash;protein correlation driven by post-transcriptional and translational regulation\u0026nbsp;(14,15). Demographic factors, particularly age, may further contribute to variability in measured VEGFA levels\u003cem\u003e\u0026nbsp;\u003c/em\u003e(16).\u003c/p\u003e\n\u003cp\u003eDespite growing interest in molecular biomarkers of preclinical neurodegeneration, little is known about peripheral \u003cem\u003eVEGFA\u003c/em\u003e expression in individuals at elevated risk of PD, particularly first-degree relatives (FDRs) (17,18). FDRs represent a key population for biomarker studies not only because of their higher lifetime risk, but also because subtle motor and non-motor abnormalities - including hyposmia, anxiety, or REM sleep behavior disorder - have been reported even in asymptomatic relatives. Such early alterations suggest underlying biological changes that precede clinical onset (19\u0026ndash;22). Experimental data further support a potential role of VEGF signaling in early neurodegenerative processes (5).\u003c/p\u003e\n\u003cp\u003eIn this study, we investigated peripheral blood \u003cem\u003eVEGFA\u003c/em\u003e expression in patients with PD, their asymptomatic first-degree relatives, and healthy controls. We also examined age-related expression patterns to determine whether \u003cem\u003eVEGFA\u003c/em\u003e may serve as an early biomarker of neurodegeneration in at-risk individuals.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cem\u003eParticipants and study design\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePatients with a clinically confirmed diagnosis of Parkinson\u0026rsquo;s disease (PD) were recruited according to the Movement Disorder Society (MDS) diagnostic criteria (23). Disease severity was evaluated using the Movement Disorder Society\u0026ndash;sponsored revision of the Unified Parkinson\u0026rsquo;s Disease Rating Scale (MDS-UPDRS) and the modified Hoehn and Yahr scale (24). Asymptomatic first-degree relatives (FDRs), defined as siblings or children of PD patients, with no history of neurological disease were included. Healthy controls had no personal or family history of PD or other neurological disorders. Inclusion and exclusion criteria for all study groups are listed in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSample collection and processing\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePeripheral venous blood samples were collected into K₃EDTA tubes (SARSTEDT AG \u0026amp; Co. KG, N\u0026uuml;mbrecht, Germany) and immediately stored at \u0026minus;80\u0026deg;C until processing to minimize RNA degradation. After thawing, total RNA was isolated from 200 \u0026micro;L of frozen whole blood using the NucleoSpin\u0026reg; RNA Blood Kit (MACHEREY-NAGEL GmbH \u0026amp; Co. KG, D\u0026uuml;ren, Germany) according to the manufacturer\u0026rsquo;s instructions. RNA purity and quantity were determined spectrophotometrically using a NanoDrop 2000 instrument (Thermo Fisher Scientific, Waltham, MA, USA). Samples with an A260/280 ratio outside the 1.8\u0026ndash;2.0 range were excluded.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eRT-qPCR analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eQuantification of \u003cem\u003eVEGFA\u003c/em\u003e expression was performed using reverse transcription quantitative polymerase chain reaction (RT-qPCR) with the KAPA PROBE FAST Universal One-Step qRT-PCR Master Mix (2X) Kit (Roche Diagnostics GmbH, Mannheim, Germany). TaqMan probes were used for the target gene \u003cem\u003eVEGFA\u003c/em\u003e (Assay ID: Hs03929054_s1) and the reference gene \u003cem\u003eB2M\u003c/em\u003e (Assay ID: Hs00984230_m1).\u003c/p\u003e\n\u003cp\u003eEach reaction had a total volume of 20 \u0026micro;L, including 2 \u0026micro;L of isolated RNA. Amplification was performed on a qTOWER\u0026sup3; touch/qTOWER\u0026sup3; G touch Real-Time PCR Thermal Cycler (Analytik Jena GmbH+Co. KG, Jena, Germany) using qPCRsoft 4.1 software.\u003c/p\u003e\n\u003cp\u003eThe thermal cycling protocol consisted of:\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003ereverse transcription at 42 \u0026deg;C for 5 min,\u003c/li\u003e\n \u003cli\u003epolymerase activation at 95 \u0026deg;C for 3 min,\u003c/li\u003e\n \u003cli\u003efollowed by 40 cycles of 95 \u0026deg;C for 3 s and 60 \u0026deg;C for 30 s.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eEach sample was analyzed in two to four technical replicates. Samples with a Ct standard deviation \u0026gt; 0.25 across replicates were excluded.\u003c/p\u003e\n\u003cp\u003eIn this study, \u0026lsquo;\u003cem\u003eVEGFA\u003c/em\u003e expression\u0026rdquo; denotes peripheral blood mRNA quantified by one-step RT-qPCR and normalized to the reference gene\u0026nbsp;\u003cem\u003eB2M\u003c/em\u003e. The results were expressed as the relative expression of \u003cem\u003eVEGFA\u003c/em\u003e to \u003cem\u003eB2M\u003c/em\u003e, calculated using the 2^-\u0026Delta;\u0026Delta;Ct method (25,26). All relative expression values were natural-log transformed (logVEGFA) for subsequent analysis.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFor each participant, the geometric mean of the technical replicates of relative \u003cem\u003eVEGFA\u003c/em\u003e expression was calculated (due to proportional data). These values were then\u0026nbsp;\u003cstrong\u003enatural-log transformed (the variable logVEGFA)\u003c/strong\u003e to follow normal distribution as confirmed by the\u0026nbsp;\u003cstrong\u003eShapiro\u0026ndash;Wilk\u003c/strong\u003e test. Subsequent analyses used logVEGFA as specified below.\u003c/p\u003e\n\u003cp\u003eThe baseline characteristics were analyzed using descriptive statistics. Continuous variables are presented as the mean \u0026plusmn; standard deviation, categorical variables as absolute and relative frequencies (%). Between-group differences for continuous variables were analyzed with the analysis of variance (ANOVA) with the Games-Howell or Tukey post-hoc test. Differences between groups for categorical variables were assessed using the chi-square test or Fisher\u0026rsquo;s exact test, as appropriate.\u003c/p\u003e\n\u003cp\u003eThe correlation between age and logVEGFA was assessed using Pearson\u0026rsquo;s correlation coefficient. Receiver operating characteristic (ROC) analysis was performed to determine the optimal cut-off value for \u003cem\u003eVEGFA\u003c/em\u003e expression distinguishing PD patients. The cut-off was selected by maximizing Youden\u0026rsquo;s index.\u003c/p\u003e\n\u003cp\u003eAll statistical analyses were performed using IBM SPSS 30.0 statistical software. Two-tailed p-values \u0026lt; 0.05 were considered statistically significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParticipants were recruited between January 2023 and December 2024 at the Faculty of Medicine, University of Ostrava, and collaborating neurology outpatient clinics. The study was approved by the Ethics Committee of the Faculty of Medicine, University of Ostrava (Approval No. SGS02/LF/2023) and conducted in accordance with the Declaration of Helsinki (2013 revision). Written informed consent was obtained from all participants. Data processing complied with the EU General Data Protection Regulation (GDPR, 2016/679).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 123 subjects (64 males, mean age: 57.5 \u0026plusmn; 14.6 years) were enrolled in the study: 41 patients with clinically established PD (29 males; mean age: 66.3 \u0026plusmn; 9.6 years), 52 asymptomatic FDRs (22 males; mean age: 50.2 \u0026plusmn; 13.9 years), and 30 healthy controls (13 males, mean age: 58.1 \u0026plusmn; 14.8 years). All details pertaining to the studied sample are available in Table 2. Given the observed correlation with age and statistically significant differences between the groups, the cohort was subsequently stratified into two age categories: \u0026lt; 60 and \u0026gt; 60 years.\u003c/p\u003e\n\u003cp\u003eComparison of logVEGFA values across all groups revealed significant differences (p = 0.004, ANOVA Welch test), with the lowest expression observed in the group of healthy controls (Table 2). Post hoc testing using the Games-Howell test showed significant differences between healthy controls and patients with PD (p = 0.003), as well as between healthy controls and FDRs (p = 0.011). No significant differences were observed between patients with PD and their relatives (p = 0.773; Table 3, Figure 1a).\u003c/p\u003e\n\u003cp\u003eWhen analyzing age-stratified subgroups, differences in logVEGFA expression between groups persisted only in younger participants (p \u0026lt; 0.001), whereas they were no longer statistically significant among older individuals (p = 0.643; Tables 2 and 3, Figure 1b).\u003c/p\u003e\n\u003cp\u003eThe healthy control group exhibited a moderate linear trend of increasing logVEGFA with age (Pearson\u0026rsquo;s correlation coefficient r = 0.571, p \u0026lt; 0.001), whereas no such significant correlation was observed in patients with PD or FDRs (Figure 2).\u003c/p\u003e\n\u003cp\u003eBased on the previous results, ROC analysis was conducted to assess the diagnostic performance of logVEGFA (i.e., relative \u003cem\u003eVEGFA\u003c/em\u003e expression) in distinguishing patients with PD from healthy controls. In participants aged \u0026le; 60 years, where the differences between groups were most pronounced, \u003cem\u003eVEGFA\u003c/em\u003e expression (logVEGFA) showed an excellent ability to distinguish patients with PD from healthy individuals. The area under the ROC curve (AUROC) reached 0.897 (95% confidence interval: 0.771\u0026ndash;1.000, p \u0026lt; 0.001). According to the maximal Youden\u0026rsquo;s index, the optimal cut-off value was determined to be logVEGFA = \u0026ndash;1.346 (i.e., 0.260 for relative expression \u003cem\u003eVEGFA\u003c/em\u003e), with 100% sensitivity, 73.3 % specificity, and an overall accuracy of 84.6% (Figure 3).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we demonstrated that peripheral \u003cem\u003eVEGFA\u003c/em\u003e expression is elevated not only in patients with PD but also in asymptomatic FDRs compared with healthy controls (Tables 2 and 3). Age-stratified analysis revealed that these group differences in\u0026nbsp;\u003cem\u003eVEGFA\u003c/em\u003e expression were most pronounced in individuals aged \u0026le; 60 years.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn healthy controls, age-related decline is well documented for several non-motor domains relevant to prodromal PD, including olfaction and sleep (27\u0026ndash;30). In contrast, olfactory abnormalities tend to persist across age strata in PD and at-risk cohorts (31,32). We observed an age-associated increase in peripheral\u0026nbsp;\u003cem\u003eVEGFA\u003c/em\u003e expression exclusively among healthy controls, a trend consistent with age-linked low-grade inflammation and leukocyte compositional changes rather than endothelial expression shifts\u0026nbsp;(33). The absence of this association in PD patients and FDRs suggests that early neurodegenerative processes may interfere with physiological, age-associated compensatory mechanisms. These findings underscore the importance of incorporating age stratification when evaluating molecular biomarkers in prodromal or early PD\u0026nbsp;(32,34).\u003c/p\u003e\n\u003cp\u003ePrior studies assessing circulating VEGF-A protein levels have shown heterogeneous results, with several reporting no significant differences between PD patients and controls (13). \u0026nbsp;Such variability may reflect heterogeneity in cohorts, methodological differences, or the inherently limited correlation between mRNA expression and protein abundances due to post-transcriptional and translational regulation. Our study specifically focused on\u0026nbsp;\u003cem\u003eVEGFA\u003c/em\u003e transcript levels in a relatively younger population (\u0026le; 60 years), which may yield better sensitivity for detecting early disease-related molecular changes.\u003c/p\u003e\n\u003cp\u003eThe dual role of VEGF-A in the central nervous system adds biological plausibility to these findings. VEGF-A supports angiogenesis, enhances regional cerebral blood flow, and promotes dopaminergic neuron survival, all of which have demonstrated protective effects in experimental PD models (5). Conversely, dysregulated VEGF-A signaling has been linked to blood-brain barrier dysfunction and neuroinflammatory processes implicated in PD pathogenesis (3,12). Whether the elevated \u003cem\u003eVEGFA\u003c/em\u003e expression observed in FDRs represents an adaptive compensatory response or an early molecular hallmark of neurodegeneration remains unclear.\u003c/p\u003e\n\u003cp\u003eFDRs constitute a biologically relevant at-risk population due to their increased lifetime risk and their tendency to exhibit subtle motor and non-motor abnormalities - such as hyposmia, anxiety, depression, or REM sleep behavior disorder - even in the absence of clinically manifest PD (19\u0026ndash;22). Imaging studies further support early biological alterations, with dopaminergic deficits on DAT-SPECT reported in hyposmic relatives of PD patients (22,34). Family history also modifies disease risk, as siblings of patients with early-onset PD are more likely to develop PD and may present at a younger age (35).\u003c/p\u003e\n\u003cp\u003eElevated peripheral\u0026nbsp;\u003cem\u003eVEGFA\u003c/em\u003e expression in FDRs, as observed in our study, raises the question of whether this pattern reflects an adaptive compensatory response or an early molecular component of the pathogenic process. A common genetic variant in\u0026nbsp;\u003cem\u003eVEGFA\u003c/em\u003e (rs3025039) has been identified as a risk factor for sporadic PD (5), suggesting that genetic susceptibility and regulatory dynamics of\u0026nbsp;\u003cem\u003eVEGFA\u003c/em\u003e expression may influence vulnerability to neurodegeneration.\u003c/p\u003e\n\u003cp\u003eFrom a biomarker perspective, our findings suggest that peripheral\u0026nbsp;\u003cem\u003eVEGFA\u003c/em\u003e expression, particularly when integrated with established prodromal markers such as hyposmia or REM sleep behavior disorder and with probability-based risk algorithms, may contribute to improved risk stratification. \u0026nbsp;Previous studies have reported increased VEGF-A or VEGFR-1 levels in dopaminergic neurons and astrocytes of the substantia nigra (5,11), as well as elevated VEGF in cerebrospinal fluid (12). Our results extend these observations by demonstrating that asymptomatic FDRs also show elevated peripheral\u0026nbsp;\u003cem\u003eVEGFA\u003c/em\u003e levels, potentially reflecting either early compensatory activation of VEGF signaling or an early step in disease evolution.\u003c/p\u003e\n\u003cp\u003eOur findings also suggest that peripheral \u003cem\u003eVEGFA\u003c/em\u003e expression has potential utility as an early biomarker of PD risk. In participants\u0026nbsp;\u003cstrong\u003eaged \u0026le; 60 years\u003c/strong\u003e, we identified a \u003cem\u003eVEGFA\u003c/em\u003e expression threshold (logVEGFA = \u0026ndash;1.346) that distinguished patients with PD from healthy controls with high accuracy (sensitivity, 100%; specificity, ~73.3%). Because peripheral expression can be influenced by systemic variables, this biomarker is likely to be most informative when combined with additional prodromal markers or biological measures. Future longitudinal studies are needed to determine whether elevated\u0026nbsp;\u003cem\u003eVEGFA\u003c/em\u003e expression in at-risk individuals predicts conversion to PD and whether interventions - such as exercise or neuroprotective strategies - may modify VEGFA expression and alter disease trajectory.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStudy limitations\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis study has several limitations that should be considered when interpreting the results. First, the age distribution of the cohort may have influenced the findings, as early-onset PD (\u0026le; 50 years) is relatively rare, and only a small proportion of patients with PD are diagnosed before the age of 60 (36,37). This may amplify age-related effects in subgroup analyses.\u003c/p\u003e\n\u003cp\u003eSecond, the logVEGFA values reflect relative gene expression normalized to \u003cem\u003eB2M\u003c/em\u003e rather than circulating VEGF-A protein levels; given that mRNA\u0026ndash;protein correlations are typically low and gene-specific, transcript levels may not directly reflect systemic protein concentrations (14). As an additional methodological note, RT-qPCR data were normalized to a single reference gene, \u003cem\u003eB2M\u003c/em\u003e. Although \u003cem\u003eB2M\u003c/em\u003e showed stable expression in our dataset, the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines recommend using at least two validated reference genes to minimize normalization bias.\u003c/p\u003e\n\u003cp\u003eFinally, the genetic analysis was not comprehensive because no systematic screening was performed for known PD-related mutations. Although monogenic forms represent only approximately 5\u0026ndash;10% of all PD cases and occur more frequently in early-onset forms, testing a broader panel of genes, such as \u003cem\u003eLRRK2\u003c/em\u003e, \u003cem\u003ePRKN\u003c/em\u003e, \u003cem\u003ePINK1\u003c/em\u003e, or \u003cem\u003eDJ1\u003c/em\u003e, could have provided further insight into the relationship between genetic predisposition and \u003cem\u003eVEGFA\u003c/em\u003e expression (38\u0026ndash;40).\u003c/p\u003e\n\u003cp\u003eDespite these limitations, the principal finding remains: both PD patients and their first-degree relatives exhibit elevated peripheral\u0026nbsp;\u003cem\u003eVEGFA\u003c/em\u003e expression compared with healthy controls. Taken together with age-stratified analyses and ROC results, these findings provide preliminary support for\u0026nbsp;\u003cem\u003eVEGFA\u003c/em\u003e as a candidate biomarker for early PD risk.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003ePeripheral \u003cem\u003eVEGFA\u003c/em\u003e expression was significantly higher in both patients with PD and their first-degree relatives compared with healthy controls, with the clearest group differences observed in individuals aged \u0026le; 60 years. Only healthy controls showed an age-related increase in \u003cem\u003eVEGFA\u003c/em\u003e expression, suggesting disruption of physiological regulation in PD and at-risk individuals. In younger participants, \u003cem\u003eVEGFA\u003c/em\u003e expression demonstrated excellent discriminatory ability, supporting its potential utility as an early biomarker when combined with other prodromal measures. Longitudinal studies are needed to determine its predictive value for conversion to PD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all participants for their involvement in this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Institute for Neurological Research, Programme EXCELES (Project No. LX22NPO5107), funded by the European Union \u0026ndash; Next Generation EU, and by the University of Ostrava (Grant SGS02/LFOU/2023). The funders had no role in study design, data collection, data analysis, data interpretation, or manuscript preparation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompetig Interests.\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions.\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEA, DSk and PB conceived and designed the study. EA, FS and TM collected clinical data and samples. KTS, KF and AK performed laboratory analyses. EA and DSa conducted statistical analyses. EA, ZH, PD and DSa drafted the manuscript. All authors contributed to data interpretation, critically revised the manuscript, and approved the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement.\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCalvo PM, Hern\u0026aacute;ndez RG, de la Cruz RR, et al. Role of vascular endothelial growth factor as a critical neurotrophic factor for the survival and physiology of motoneurons. \u003cem\u003eNeural Regen Res\u003c/em\u003e 2022; 18: 1691\u0026ndash;1696.\u003c/li\u003e\n\u003cli\u003eG\u0026oacute;ra-Kupilas K, Jośko J. Review article The neuroprotective function of vascular endothelial growth factor (VEGF). \u003cem\u003eFolia Neuropathol\u003c/em\u003e 2005; 43: 31\u0026ndash;39.\u003c/li\u003e\n\u003cli\u003eShim JW, Madsen JR. 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Risk factors for Parkinson\u0026rsquo;s disease and impaired olfaction in relatives of patients with Parkinson\u0026rsquo;s disease. \u003cem\u003eMovement Disorders\u003c/em\u003e 2007; 22: 2249\u0026ndash;2255.\u003c/li\u003e\n\u003cli\u003ePostuma RB, Berg D, Stern M, et al. MDS clinical diagnostic criteria for Parkinson\u0026rsquo;s disease. \u003cem\u003eMovement Disorders\u003c/em\u003e 2015; 30: 1591\u0026ndash;1601.\u003c/li\u003e\n\u003cli\u003eGoetz CG, Tilley BC, Shaftman SR, et al. Movement Disorder Society-sponsored revision of the Unified Parkinson\u0026rsquo;s Disease Rating Scale (MDS-UPDRS): Scale presentation and clinimetric testing results. \u003cem\u003eMovement Disorders\u003c/em\u003e 2008; 23: 2129\u0026ndash;2170.\u003c/li\u003e\n\u003cli\u003eLivak KJ, Schmittgen TD. 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Monogenic Parkinson\u0026rsquo;s Disease: Genotype, Phenotype, Pathophysiology, and Genetic Testing. \u003cem\u003eGenes (Basel)\u003c/em\u003e 2022; 13: 471.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e \u003cstrong\u003eInclusion and exclusion criteria for subjects enrolled to the prospective study\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"598\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eStudy group\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 165px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eInclusion criteria\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 324px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eExclusion criteria\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePatients with Parkinson\u0026acute;s disease\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 165px;\"\u003e\n \u003cp\u003eClinically established PD (Postuma RB et al 2015)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 324px;\"\u003e\n \u003cp\u003eDiagnosed neurological or psychiatric disease except PD; known genetic mutation associated with PD; oncological treatment, ongoing infection or current tretment with immunosuppressants or corticosteroids\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 165px;\"\u003e\n \u003cp\u003eMale or female\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 324px;\"\u003e\n \u003cp\u003eKnown impairment of olfactory function (other than PD etiology)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 165px;\"\u003e\n \u003cp\u003eAge \u0026ge; 20 years\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 324px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePD First-degree relative\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 165px;\"\u003e\n \u003cp\u003eMale or female\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 324px;\"\u003e\n \u003cp\u003eDiagnosed neurological or psychiatric disease; known genetic mutation associated with PD; oncological treatment, ongoing infection or current tretment with immunosuppressants or corticosteroids\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 165px;\"\u003e\n \u003cp\u003eAge \u0026ge; 20 years\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 324px;\"\u003e\n \u003cp\u003eKnown impairment of olfactory functions\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 165px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 324px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHealthy subjects\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 165px;\"\u003e\n \u003cp\u003eSubject without any known chronic disease\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 324px;\"\u003e\n \u003cp\u003eDiagnosed neurological or psychiatric disease; oncological treatment, ongoing infection or current tretment with immunosuppressants or corticosteroids\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 165px;\"\u003e\n \u003cp\u003eMale or female\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 324px;\"\u003e\n \u003cp\u003eKnown impairment of olfactory functions\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 165px;\"\u003e\n \u003cp\u003eAge \u0026ge; 20 years\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 324px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003ePD \u0026ndash; Parkinson\u0026acute;s disease\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Baseline patient characteristics.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRelatives\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControls\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003evalue\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAll participants\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of participants; n\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge; mean\u003c/strong\u003e\u003cstrong\u003e\u0026plusmn;SD (years)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e66.3\u0026nbsp;\u0026plusmn; 9.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e50.2\u0026nbsp;\u0026plusmn; 13.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e58.1\u0026nbsp;\u0026plusmn; 14.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMale sex; n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e29 (70.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e22 (42.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e13 (43.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e0.013\u003csup\u003e\u0026nbsp;b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003cstrong\u003elogVEGFA; mean\u003c/strong\u003e\u003cstrong\u003e\u0026plusmn;SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e-0.514\u0026nbsp;\u0026plusmn; 0.590\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e-0.605\u0026nbsp;\u0026plusmn; 0.687\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e-1.240\u0026nbsp;\u0026plusmn; 1.024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e0.004\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge \u0026le; 60 years\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of participants; n\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge; mean\u003c/strong\u003e\u003cstrong\u003e\u0026plusmn;SD (years)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e53.9 \u0026plusmn; 6.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e45.8 \u0026plusmn; 11.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e45.5 \u0026plusmn; 9.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e0.020\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMale sex; n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e7 (63.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e19 (46.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e6 (40.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e0.471\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003cstrong\u003elogVEGFA; mean\u003c/strong\u003e\u003cstrong\u003e\u0026plusmn;SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e-0.316\u0026nbsp;\u0026plusmn; 0.534\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e-0.637 \u0026plusmn; 0.703\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e-1.717\u0026nbsp;\u0026plusmn; 0.991\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge \u0026gt; 60 years\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of participants; n\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge; mean\u003c/strong\u003e\u003cstrong\u003e\u0026plusmn;SD (years)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e70.8\u0026nbsp;\u0026plusmn; 6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e66.6\u0026nbsp;\u0026plusmn; 6.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e70.7\u0026nbsp;\u0026plusmn; 4.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e0.067\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMale sex; n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e22 (73.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e3 (27.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e7 (46.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e0.019\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003cstrong\u003elogVEGFA; mean\u003c/strong\u003e\u003cstrong\u003e\u0026plusmn;SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e-0.587\u0026nbsp;\u0026plusmn; 0.601\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e-0.484\u0026nbsp;\u0026plusmn; 0.642\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 21px;\"\u003e\n \u003cp\u003e-0.762\u0026nbsp;\u0026plusmn; 0.838\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e0.643\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e Kruskal-Wallis test; \u003csup\u003eb\u003c/sup\u003e Chi-square test; \u003csup\u003ec\u003c/sup\u003e ANOVA (the Welch test); PD \u0026ndash; Parkinson\u0026acute;s disease; SD \u0026ndash; standard deviation; VEGFA \u0026ndash; vascular endothelial growth factor isoform A\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Pairwise comparisons of logVEGFA between groups (all participants and separately participats\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026le;\u003c/strong\u003e\u003cstrong\u003e60 years).\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"595\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 255px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAll participants\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge \u0026le; 60 years\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 255px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ep\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-value \u003csup\u003ea\u003c/sup\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ep\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-value \u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 255px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHealthy controls vs. PD patients\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 170px;\"\u003e\n \u003cp\u003e0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 170px;\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 255px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHealthy controls vs. PD relatives\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 170px;\"\u003e\n \u003cp\u003e0.011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 170px;\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 255px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePD patients vs. PD relatives\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 170px;\"\u003e\n \u003cp\u003e0.773\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 170px;\"\u003e\n \u003cp\u003e0.426\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003ePD \u0026ndash; Parkinson\u0026rsquo;s disease, \u003csup\u003ea\u0026nbsp;\u003c/sup\u003eadjusted by the Games-Howell post-hoc test, \u003csup\u003eb\u003c/sup\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eadjusted by the Tukey post-hoc test\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Parkinson’s disease, VEGFA, gene expression, biomarkers, first-degree relatives, RT-qPCR","lastPublishedDoi":"10.21203/rs.3.rs-8433576/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8433576/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Vascular endothelial growth factor A (VEGFA) contributes to angiogenesis and exerts neurotrophic and neuroprotective effects. Altered \u003cem\u003eVEGFA\u003c/em\u003e expression has been implicated in neurodegenerative processes, including Parkinson´s disease (PD), yet findings from peripheral transcriptomic studies remain inconsistent. Data on individuals at increased risk, particularly first-degree relatives (FDRs), are limited.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e To evaluate peripheral blood \u003cem\u003eVEGFA\u003c/em\u003e mRNA expression in patients with PD and their asymptomatic FDRs compared with healthy controls, and to assess age-related patterns and diagnostic performance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Peripheral blood \u003cem\u003eVEGFA\u003c/em\u003eexpression was analyzed in 123 participants (PD patients, FDRs, healthy controls) using one-step RT-qPCR normalized to \u003cem\u003eB2M\u003c/em\u003e. Relative expression values were log-transformed (logVEGFA). Group differences, age correlations, and diagnostic discrimination were assessed using ANOVA, Pearson correlation, and receiver operating characteristic (ROC) analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Both PD patients and FDRs showed significantly higher logVEGFA levels than healthy controls (p = 0.004), with the most pronounced differences observed in participants aged ≤ 60 years (p \u0026lt; 0.001). A positive correlation between age and logVEGFA was detected only in healthy controls. In participants aged ≤ 60 years, logVEGFA demonstrated excellent discrimination between PD patients and healthy individuals (AUC 0.897; 95% CI 0.771–1.000; sensitivity 100%, specificity 73.3%).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e Peripheral \u003cem\u003eVEGFA\u003c/em\u003e expression is elevated in both PD patients and FDRs, particularly in younger individuals. Age-stratified \u003cem\u003eVEGFA\u003c/em\u003e assessment may support early identification of individuals at risk of PD and complement existing prodromal biomarkers, pending longitudinal validation.\u003c/p\u003e","manuscriptTitle":"VEGFA Gene Expression Levels in Patients with Parkinson’s Disease and First-Degree Relatives","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-19 16:11:13","doi":"10.21203/rs.3.rs-8433576/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"34682076026849445518994996662158355620","date":"2026-05-16T16:11:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-24T16:22:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"231482689126727465678363548628243851390","date":"2026-03-17T12:39:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-16T19:54:24+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-30T17:48:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-27T00:49:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-27T00:48:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-12-23T12:00:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8c5647cc-000e-4d14-9e94-bbdc38571df0","owner":[],"postedDate":"March 19th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"34682076026849445518994996662158355620","date":"2026-05-16T16:11:15+00:00","index":117,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":64750239,"name":"Health sciences/Biomarkers"},{"id":64750240,"name":"Health sciences/Diseases"},{"id":64750241,"name":"Health sciences/Neurology"},{"id":64750242,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2026-03-19T16:11:13+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-19 16:11:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8433576","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8433576","identity":"rs-8433576","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}