SNCA locus Triplication Drives Severe Parkinson’s Disease: Platelet and Blood Expression Profiles Evidence Systemic Involvement

SNCA locus Triplication Drives Severe Parkinson’s Disease: Platelet and Blood Expression Profiles Evidence Systemic Involvement | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article SNCA locus Triplication Drives Severe Parkinson’s Disease: Platelet and Blood Expression Profiles Evidence Systemic Involvement Susanna Jiménez, Jorge Mena, David Adamuz, Abril Tuset, Mar Mallo, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8422817/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Parkinson disease (PD) is increasingly recognized as a multisystem condition with substantial immune involvement. Functional parallels described between neurons and platelets have raised interest in the latter as a window into neurodegenerative disease mechanisms. While most PD cases are sporadic, rarely, mutations or gene dosage increases of SNCA cause familial PD. We report the case of the ninth PD patient carrying a SNCA locus triplication on chromosome 4q21.1 (SNCA3x), characterized by early-onset dementia and encompassing the largest number of coding genes described to date. To explore molecular correlates of disease progression, we longitudinally profiled the gene expression in blood and platelets from the SNCA3x PD patient in early and advanced stages, compared with healthy controls and early-onset PD patients stratified by age and sex. Early-stage PD was characterized by the downregulation of 14-3-3 and monoamine transporter genes, along with the upregulation of ribosomal genes in platelets, SPP1 tv1 in blood, and of inflammatory and PPM1K phosphatase genes in both blood and platelets. In the advanced stage of PD, we observed overexpression of SNCA and MMRN1 in blood and platelets. These dynamic shifts suggest a stage-dependent platelet signature that mirrors neurodegenerative and immune pathways. Our findings reveal shared molecular mechanisms with other forms of PD and underscore platelets as a peripheral source of biomarker discovery in PD. Health sciences/Biomarkers Health sciences/Diseases Biological sciences/Genetics Health sciences/Neurology Biological sciences/Neuroscience Parkinson’s disease SNCA locus triplication gene expression platelets 14-3-3 inflammation peripheral biomarker Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Parkinson disease (PD) is one of the most common neurodegenerative movement disorders, pathologically defined by abnormal aggregation of alpha-synuclein (AS), progressive loss of dopaminergic neurons in the substantia nigra, and chronic neuroinflammation 1 . Although its exact underlying mechanisms remain incompletely understood, aberrant protein homeostasis, oxidative stress, mitochondrial dysfunction, DNA damage, impaired neurotrophic signaling, and sustained inflammatory responses have all been implicated in disease pathogenesis. Increasing evidence now positions PD as a multisystem disorder characterized by widespread immune dysregulation and systemic inflammation 2 . Peripheral inflammatory mediators such as platelets (PLTs), have emerged as a successful model system for studying PD pathoprogression 3 . PLTs share notable similarities with neurons in their degranulation/secretion patterns, vesicular trafficking, and surface receptor expression 4 . They also store and release neurotransmitters and several neuron-specific proteins, including amyloid precursor protein (APP), β-amyloid (Aβ), tau protein, and brain-derived neurotrophic factor (BDNF) 5 . Remarkably, PLTs contain the highest concentration of AS per milligram of cellular protein in the blood 6 and express several proteins related to PD 7–9 . Collectively, these features position PLTs as a suitable peripheral model for PD research. Despite this, the role of peripheral inflammation in the progression of PD clinical symptoms remains unclear. Increasing evidence supports a multifactorial origin of PD, with the immune system positioned at the crossroads of genetic and environmental interactions that influence disease risk 10 . Although most cases are classified as sporadic with an unknown etiology, several risk factors – including genetic predisposition – have been proposed 11 , 12 . In this regard, point mutations, as well as duplications and triplications of the alpha-synuclein ( SNCA ) gene, cause autosomal dominant familial PD. Affected carriers show clinical and pathological features of PD, and often PD with dementia (PDD) or dementia with Lewy bodies (DLB). However, whereas SNCA duplications can be non-fully penetrant, SNCA triplications are invariably associated with a rapidly progressive and aggressive PD phenotype 13 . These observations demonstrate that both toxic gain-of-function from mutant protein and overproduction of wild-type protein promote AS aggregate formation 14 . To date, only six families with SNCA triplication 14 – 19 and two apparently isolated patients 20 , 21 have been reported. The present case represents the ninth patient worldwide with SNCA locus triplication, and the third reported isolated case. Previous triplication cases have described lengths of 0.351 Mb 20 , 1.2 Mb 15 and 2.61–2.64 Mb 18 . A meta-analysis, including unpublished triplication cases, reported that the size of the SNCA locus multiplication, both triplications and homozygous duplication, ranges between 2.14 ± 1.83 Mb 13 . Here, we aimed to investigate the molecular mechanisms underlying the onset of PD and its rapid progression to PD with dementia, based on a novel case of SNCA triplication (SNCA3x). Using whole blood and PLT samples, we assessed whether immune- and inflammation-related pathways change during disease progression. In addition, we examined whether PLTs reflect molecular alterations typically observed in the brains of patients with PD. Therefore, we analyzed gene expression levels of genes located within the triplicated locus on chromosome 4q21.1, genes involved in inflammatory pathways, genes encoding 14-3-3 proteins, ribosomal genes, and the vesicular monoamine transporter gene VMAT2 ( SLC18A2 ) in both blood and PLTs. MATERIALS AND METHODS Participants A patient carrying an SNCA locus triplication (SNCA3x), with blood samples collected at two time points during the disease, was included. The first sample was obtained at age 41, corresponding to de novo early-stage PD (EPD), and the second, at 44 years, when the patient presented advanced PD with dementia (PDD), after 3 years of disease duration. For comparison purposes, two additional cohorts were included. First, 15 age- and sex-matched early-onset PD patients, diagnosed according to the MDS clinical diagnostic criteria for Parkinson’s disease 22 , were prospectively recruited during consultations at the Hospital Universitari Germans Trias i Pujol (Badalona, Spain). Second, 18 age- and sex-matched volunteers (CTRLs) without a family history of neurodegenerative disease were recruited as healthy controls at the Research Institute Germans Trias i Pujol (Badalona, Spain). The study was approved by the Clinical Research Ethics Committee of the University Hospital Germans Trias i Pujol (Date: February 25, 2022; No. PI-22-024), and all participants provided informed consent in accordance with the Declaration of Helsinki. Genetic testing and copy number analyses Genomic DNA was purified from whole blood using standard methods. Genetic testing was performed using an in-house diagnostic panel for parkinsonism and dystonia (PD_DYT_2022_v2, IAD223176, and spike in IAD223184; Thermo Fisher Scientific, Waltham, MA, USA) and targeted NGS gene sequencing (Ion GeneStudio S5 System; Thermo Fisher Scientific). The genes included in the panel are shown in Table S1 . Multiplex ligation-dependent probe amplification (MLPA) was performed to detect copy-number variations in PD-associated genes (PARK2, SNCA, PINK1, PARK7, ATP13A2) using the SALSA MLPA Parkinson Probemix 1 (MRC Holland, Amsterdam, the Netherlands), according to the manufacturer's instructions. The size of the triplicated region on chromosome 4q22.1 was determined by the CytoScan High Density Array (CytoScan HD Array, Applied Biosystems, Thermo Fisher Scientific), containing more than 2.6 million copy number markers and was processed on the GeneChip System 3000. Data was analyzed with the Chromosome Analysis Suite (ChAS) software. Gene selection for expression analyses Among the genes located within the triplicated SNCA locus, seven genes ( SPP1 , ABCG2 , PPM1K , HERC6 , GPRIN3 , SNCA , MMRN1 ) with relevance to neurodegenerative processes were selected. Additionally, eleven genes with established links to PD neuropathogenesis were also included: inflammatory pathway genes ( NLRP3 , TLR2 , TLR4 ); 14-3-3 protein–encoding genes ( YWHAQ , YWHAZ , YWHAB , YWHAG , YWHAH ); ribosomal genes ( RPS18 , RPL11 ); and the vesicular monoamine transporter gene SLC18A2 (VMAT2). RNA isolation and reverse transcription RNA from whole blood was extracted using the PAXGene Blood RNA Kit (PreAnalytiX, Hombrechtikon, Switzerland; Cat. no. 763134), and RNA from PLTs was extracted using the mirVANA miRNA Isolation Kit (Thermo Fisher Scientific; Cat. no. AM1561), following the manufacturer’s protocols. RNA concentration and purity were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific), with A260/A280 ratios adjusted to 1.8–2.0, noting that PLTs-derived RNA may yield slightly lower values. Reverse transcription was performed using 1 µg of total RNA from blood and 200 ng from PLTs with Ready-to-go™ You-Prime First-Strand Beads (Cytiva, Marlborough, MA, USA; Cat. no. 2796401). Primer design and real-time PCR Transcript variant (tv)-specific forward primers targeted SNCA exons 2a, 2b, or 1 for SNCA tv1, SNCA tv2, and SNCA tv3, respectively, with a common reverse primer in exon 4. Primer sequences and combinations are provided in Table S2 . Relative gene expression was assessed in blood and PLT samples by qPCR (LightCycler 480 I; Roche, Penzberg, Germany) using LUNA Universal qPCR Master Mix (New England Biolabs, Ipswich, MA, USA) in 14 µL reactions containing 1 µL cDNA. Reference genes were PBGD1 and ACTB for blood, and RDX and SNRDP3 for PLTs 23 . PD and CTRL samples were run in duplicate; samples corresponding to the two extractions from SNCA3x were run in quadruplicate. Standard curves for target and reference genes were generated in each run using serial dilutions of a control cDNA sample. Relative expression was calculated using the geometric averaging of multiple internal control genes 24 . Statistical analysis Analyses were performed using RStudio (4.3.2) and BioRender Graph software. Relative gene expression was calculated using the control group as the baseline for PD and the EPD group as the baseline for PDD. Data are presented as means. Normality and variance homogeneity were assessed using the Shapiro–Wilk and Levene’s tests, respectively. For normally distributed data with equal variances, a two-tailed Student’s t -test was applied; for unequal variances, a two-tailed Welch’s t -test was used. For non-normally distributed data, the Mann–Whitney U test was applied. All p-values were adjusted for multiple testing using the FDR correction. Statistical significance was assessed based on FDR-adjusted p-values ≤ 0.05. The two SNCA3x time points represent repeated measures in the same individual and were not subjected to inferential testing. RESULTS Clinical description of the SNCA3x carrier A female patient showed depressive symptoms and generalized bradykinesia at age 41, with a sudden onset of symptoms after the second in vitro fertilization treatment. The initial neurological examination revealed rigidity and altered tapering in upper extremities, slow alternating movements and a gait characterized by small steps, with her right leg slightly dragging. The UPDRS III score was 32, and the Hoehn and Yahr (HY) stage was 2. Dopamine transporter imaging (SPECT) revealed severe presynaptic dopaminergic depletion, and 123I-MIBG myocardial scintigraphy showed absent myocardial uptake, indicating postganglionic cardiac depletion. The patient was diagnosed with PD. After six months of treatment with levodopa, her symptoms improved moderately. With regards to her familial history, two paternal uncles developed parkinsonism at the ages of 55 and 75. Fifteen months after disease onset, she had memory complaints, and complained about more clumsiness of her right leg, dyskinesias, sialorrhea and wearing-off phenomenon. A neuropsychological battery revealed moderate to severe alteration of verbal and working memory, a marked slowing and increased response latency of spontaneous language. Eighteen months after disease onset, the patient experienced major functional limitation with the wearing-off phenomenon and significant nocturnal akinesia. Three months later, she complained of disturbing dyskinesias, especially in her right leg, and the wearing-off phenomenon predominantly in the morning. She developed nocturnal episodes compatible with REM sleep behavior disorder (RBD), and clonazepam 0,5 mg once a day was started. Three years after the disease onset, she was on treatment with levodopa/benserazide 200 mg tid and amantadine 100 mg tid. A follow-up neuropsychological battery showed worse scores in response speed of spontaneous language, verbal and working memory, attention, executive functions, object naming and visuospatial functions. In her last neurological assessment, 5 years after disease onset, the UPDRS III score was 23, HY stage 2, and she was under levodopa/benserazide 200 mg tid, amantadine 100 mg tid, clonazepam 0,5 mg od, entacapone 200 mg qid. She was relatively autonomous but needed some aid when walking in the street. Demographic data of PD patients and control individuals are shown in Table S3 . Genetic testing Neither known disease-causing variants nor other rare variants were found in the 92 genes tested by targeted NGS. MLPA analysis revealed a double dose of the SNCA gene, indicating the presence of either a homozygous duplication or a heterozygous triplication. To determine its origin, we analyzed samples from both parents, but neither of them carried the triplication. Thus, the patient harbored a de novo SNCA triplication. The results of the CytoScan HD Array revealed that the triplication spans 2.41 Mb and encompasses 21 genes, 19 of which are protein-coding ( Fig. S1 ). Gene expression differences in blood and PLTs are sex-dependent To understand whether gene expression in whole blood and PLTs differed between PD and CTRLs, sex was included as a covariate. In blood, SPP1 tv1, SNCA tv1 and SNCA tv2 were upregulated in PD vs. CTRLs ( SPP1 tv1: p = 0.0064; SNCA tv1: p = 0.0204; SNCA tv2: p = 0.0237) ( Fig. 1a-c ). After sex-adjusted analysis, no differences were found among females, but all three transcripts remained significantly upregulated in male PD (mPD) vs. male CTRLs (mCTRLs) ( SPP1 tv1: p = 0.00645; SNCA tv1: p = 0.0204; SNCA tv2: p = 0.0237) ( Fig. S2a-c ). Additionally, SPP1 tv1 expression was increased in female CTRLs (fCTRLs) vs. mCTRLs (p = 0.0397) ( Fig. 1d ). In PLTs, SNCA tv2 and SNCA tv3 were downregulated ( SNCA tv2: p = 0.0325; SNCA tv3: p = 0.027), and MMRN1 was upregulated (p = 0.0464) in PD ( Fig. 1e-g ), but these changes lost significance after sex stratification (adjusted p > 0.05). TLR2 and YWHAB were also downregulated in PD vs. CTRLs ( Fig. S2d-e ). When considering sex as a covariable, TLR2 , TLR4 and YWHAB showed significantly decreased expression in female PD (fPD) compared to fCTRLs ( TLR2 : p = 0.0382; TLR4 : p = 0.0187; YWHAB : p = 0.0343) ( Fig. 1h-j ), whereas only YWHAB was decreased in mPD vs. mCTRLs (p = 0.0349) ( Fig. S2f ). In addition, TLR4 was upregulated in mPD compared with fPD (p = 0.035) ( Fig. 1k ). Upregulation of ribosomal genes RPS18 and RPL11 was observed in PD vs. CTRLs ( RPS18 : p = 0.0057; RPL11 : p = 0.0015) ( Fig. 2l-m ); although, these differences remained statistically significant only in men ( RPS18 : p = 0.0219; RPL11 : p = 0.0316) ( Fig. S2g-h ). Due to the strong sex-specific effects, and the fact that the SNCA3x patient was female, subsequent comparisons between early-stage PD (EPD) and PD dementia (PDD) were performed only with fPD and fCTRL cases. Figure 1. Differentially expressed genes in PD vs CTRLs in blood and PLTs. Parkinson’s disease (PD), controls (CTRL), female PD (fPD), male PD (mPD), female controls (fCTRL) and male controls (mCTRL) (*p < 0.05, **p < 0.01). SNCA transcript expression shifts depending on PD stage Our initial results revealed marked divergence in SNCA transcript variant expression. In blood, SNCA tv3 was upregulated by 27% in PDD compared with EPD, whereas SNCA tv1 and SNCA tv2 were downregulated ( Fig. 2a ). SNCA tv3 expression was 41% higher in EPD and 64% in PDD than fCTRLs ( Fig. 2b ). By contrast, in PLTs SNCA transcript expression 214% higher in PDD than in EPD ( Fig. 2c ). Furthermore, PLT expression of SNCA tv1 was 32% lower in EPD and 77% higher in PDD, both compared with fCTRLs ( Fig. 2d ). Figure 2. SNCA transcript expression in blood and PLTs from patient SNCA3x. Early-stage PD (EPD), advanced PD with dementia (PDD), female controls (fCTRL). SPP1 tv1 is selectively overexpressed in blood during EPD SPP1 tv1 expression in blood was approximately 50% higher than the other SNCA-locus genes in EPD and 56% higher in EPD than in PDD (Fig. 3 a). In contrast, its expression was undetectable in PLTs. In EPD , MMRN1 and PPM1K expression changes are greater in PLTs than in blood In blood, the expression of the analyzed SNCA locus genes was reduced by more than 40% in PDD (Fig. 3 a), except for MMRN1 , whose expression was 50% higher than in EPD (Fig. 3 b). No statistical differences were observed between fCTRLs and EPD. However, MMRN1 expression levels were 40% higher in PDD than in fCTRLs (Fig. 3 c). In PLTs, these differences were even more pronounced with an MMRN1 expression increase of 180% in PDD vs. EPD (Fig. 3 d). In addition, its expression was 30% higher in EPD than in fCTRLs, and this difference increased to more than 100% in PDD (Fig. 3 e). PLT PPM1K expression in EPD was 150% higher than in fCTRLs (Fig. 3 f), 83% higher than in PDD and markedly elevated compared with other genes within the SNCA locus (Fig. 3 d). By contrast, PPM1K levels in EPD were comparable to those of other SNCA -locus genes in whole blood (Fig. 3 a). PLT SLC18A2 downregulation and upregulation of ribosomal genes RPS18 and RPL11 characterize EPD Since PLTs and neurons share vesicle storage features, we analyzed SLC18A2 (VMAT2) expression and the ribosomal genes RPS18 and RPL11 to further assess PLT status. In whole blood, expression levels were too low to be quantified. In PLTs, however, SLC18A2 expression was markedly reduced in EPD, 250% lower than in PDD (Fig. 4 a) and 104% lower than in fCTRLs. At the same time, no differences were observed between PDD and fCTRLs (Fig. 4 b). In contrast, RPS18 and RPL11 were upregulated in EPD by an average of 83% vs. PDD (Fig. 4 c) and 150% vs. fCTRLs (Fig. 4 d). 14-3-3 gene expression in PLTs is decreased in EPD Given the modulatory role of 14-3-3 proteins in immune responses, we quantified PLT expression of YWHAQ , YWHAG , YWHAZ , YWHAH , and YWHAB . Across disease stages, expression of 14-3-3–encoding genes was on average 300% lower in EPD compared with PDD, with the largest difference observed for YWHAB (750% lower in EPD) (Fig. 4 e). In EPD, YWHAQ , YWHAG , YWHAZ , and YWHAH were reduced on average by 75% compared with fCTRLs ( Fig. S3a ), while YWHAB was decreased by 190% vs. fCTRLs and by 170% vs. fPD (Fig. 4 f). YWHAQ , YWHAG , and YWHAZ were overexpressed by 24% in PDD compared with fCTRLs ( Fig. S3b ) and, in contrast, YWHAB was 140% lower than in fCTRLs and 46% lower than in fPD ( Fig. S3c ). Inflammatory genes are overexpressed early during PD progression To assess potential differences in inflammatory pathways between EPD and PDD, we quantified PLT and blood expression of TLR2 , TLR4 , and NLRP3 . Overall, PLT inflammatory gene expression was 67% higher in EPD than in PDD ( Fig. S4a ). Compared with fCTRLs and fPD, EPD showed reduced TLR2 expression (44% and 77%, respectively), but an increased TLR4 expression (60% and 144%, respectively) ( Fig. S4b ). By contrast, NLRP3 expression was 63% lower in PDD than in fCTRLs, and both TLR2 and TLR4 were downregulated, by 154% and 125% vs. fCTRLs, and by 75% and 27% vs. fPD ( Fig. S4c ). In blood, TLR2 , TLR4 , and NLRP3 expression was increased on average by 22% in PDD compared with EPD and reduced by 20% in EPD vs fCTRLs ( Fig. S4d ). In PDD, only a slight tendency to reduction was found compared with fCTRLs. Figure 5 summarizes the results as heatmaps, highlighting both the impaired pathways and the magnitude of change. Expression shifts were far broader in PLTs (–4.5 to 12.5) than in whole blood (–0.02 to 1.2), indicating that PLTs offer a higher discriminatory potential for detecting expression differences. Progression to advanced PD with dementia was associated with only modest expression shifts in blood, mainly a decrease in most SNCA locus genes and an increased expression of SNCA tv3, MMRN1 , and inflammatory genes. By contrast, the PLT expression profile showed a pronounced deregulation. This included a moderate increase in inflammatory genes; marked upregulation of SNCA transcripts, MMRN1 , 14-3-3 genes, and of the monoamine transporter VMAT2; as well as a moderate decrease in several SNCA -locus-associated genes ( ABCG2 , PPM1K , HERC6 , and GPRIN3 ). DISCUSSION We report patient SNCA3x, presenting a rare, early-onset, rapidly progressing form of PD caused by a SNCA locus triplication, which contains the largest number of protein-coding genes identified in an SNCA locus triplication to date. Analysis of gene expression profile across EPD and PDD stages revealed that PLTs seem to capture pathogenic expression alterations, supporting their potential as peripheral biomarkers. The analysis of the expression profiles compared with a cohort of PD patients uncovered mechanisms shared with other PD forms, establishing the case SNCA3x as a powerful model for studying molecular processes relevant to disease progression. Given their modulatory role in immune responses and functional similarities to neurons, we explored whether PLTs may serve as indicators of expression changes associated with PD pathogenesis. Functional PLT alterations, including adhesion, activation, aggregation, and degranulation, have been reported in PD patients 25 , 26 . Multimerin-1, encoded by MMRN1 , which lies adjacent to SNCA , is a PLT protein that mediates collagen-dependent adhesion. In our SNCA3x case, MMRN1 was included in the triplication, and its PLT and blood expression increased in a disease-stage-dependent manner, being higher in EPD than controls and highest in PDD. These results align with previous reports showing that SNCA locus duplications or triplications encompassing the entire MMRN1 gene are associated with dementia, whereas those that do not fully include MMRN1 show no cognitive impairment 15 , 27 , 28 . Moreover, platelet MMRN1 expression was increased in patients with PD. Emerging evidence increasingly supports a role of peripheral inflammation in the etiology of PD. A meta-analysis by Qu and colleagues 29 reported elevated levels of cytokines, including IL-6, TNF-α, CCL2, CX3CL1, and IL-β, in both blood and cerebrospinal fluid (CSF) of patients with PD, reinforcing peripheral inflammation as a hallmark of the disorder. In parallel, another study demonstrated that higher levels of systemic inflammatory markers were associated with reduced dopamine transporter (DAT) density in patients with PD 30 . Nevertheless, the extent to which peripheral inflammation contributes to PD phenotype progression remains unresolved, partly due to insufficient consideration of key modifiers, such as age of onset and sex. In our study, a significant reduction in PLT TLR2 and TLR4 expression was found in female PD patients compared with female CTRLs, and an upregulation of TLR4 in male PD patients compared with female PD patients. PLT TLR activation is known to trigger pro-inflammatory signaling cascades 31 , leading to α-granule release, and TLR2 in particular has been shown to induce PLT activation and aggregation 32 . Thus, reduced PLT TLR2 function may impair α-granule release, thereby limiting the secretion of MMRN1 and other signaling molecules. Moreover, a study by Koupenova and colleagues 33 reported higher expression levels of platelet TLR transcripts in women compared with men, suggesting that platelet TLR expression is sex-dependent. Taken together, these findings indicate that in the early stages of PD, PLTs could not only lose part of their innate immune function but also exist in a state of pre-activation, in which α-granules accumulate rather than being secreted. Importantly, the observed sex-specific differences in PLT mRNA content, including higher TLR4 levels in men with PD, support the notion that women with PD may exhibit greater dysregulation of platelet TLR expression. Nonetheless, further studies are required to delineate these sex-dependent differences and to determine their functional and clinical relevance. In addition to peripheral inflammation, our results revealed increased SPP1 tv1 expression in whole blood from male PD patients compared with controls, and in EPD compared with PDD. SPP1 acts as a cytokine in inflammation, and previous studies in AD have reported elevated SPP1 in CSF and plasma 34 . It has also been detected in plaque-associated microglia, where it has been proposed as a conserved disease-associated marker in both mice and humans 35 , 36 . Accordingly, our findings indicate elevated circulating SPP1 in PD, suggesting a novel source of this marker. These findings highlight the complexity of peripheral immune involvement and underscore the need for further analyses to elucidate not only their contribution to disease progression but also potential sex-specific differences. Accumulating data also underscore the role of 14-3-3 proteins in modulating inflammatory responses 37 – 39 . In a previous study, we detected 14-3-3 expression changes in PLTs opposite to those observed in the temporal cortex, suggesting that PLTs may capture distinct molecular alterations that mirror and inversely reflect cerebral changes 40 . Furthermore, loss of 14-3-3 function has been linked to neurodegeneration in PD 41–43 . Consistently, in PLTs, our results show a significant decrease in YWHAB expression in PD and reduced 14-3-3 expression in EPD compared with PDD. We further observed reduced PLT monoamine transporter VMAT2 ( SLC18A2 ) levels in EPD, consistent with previous reports of decreased VMAT2 in PD PLTs 44 . This observation aligns with evidence of impaired serotonin handling in PLT secretory vesicles (SVs) from patients with PD, characterized by reduced vesicular content, decreased uptake, diminished thrombin-induced release, and increased leakage 45 . In the brain, VMAT2 is thought to play a protective role by limiting cytoplasmic dopamine accumulation, and thereby reducing ROS generation and oxidative stress 45 , 46 . A similar mechanism may operate in PLTs, where reduced VMAT2 expression could foster an oxidative environment that increases cellular vulnerability. Supporting this view, we detected upregulation of PPM1K in EPD. PPM1K, a mitochondrial matrix phosphatase essential for regulating the mitochondrial permeability transition pore 47 , has been linked to mitochondrial dysfunction and neuronal loss when disrupted 48 . Notably, emerging evidence indicates functional crosstalk between peripheral tissues and the CNS in PD, with mitochondrial impairment as a potential common denominator 49 . Beyond mitochondrial alterations, PLT expression of the ribosomal genes RPS18 and RPL11 was increased in male PD patients compared with male CTRLs, and in EPD compared with PDD. Together, these findings point to a broader dysregulation of PLT molecular profiles not only across disease stages, but also in dependency on sex. Building on these findings, we investigated SNCA transcript-specific changes in blood and PLTs. While blood samples showed increased expression of SNCA tv1 and SNCA tv2 in PD, PLT expression revealed lower levels of SNCA tv2 and SNCA tv3. Moreover, between the EPD and PDD stages, transcript-specific changes diverged between blood and platelets, with all three transcripts showing increased PLT expression in PDD. Previous studies reported higher α-synuclein levels in blood from PD patients without dementia compared to those with dementia 50 , consistent with our observations. Although studies on synucleins in PLTs are scarce and sometimes inconsistent, our results position PLTs as a primary peripheral source for monitoring transcriptional changes associated with PD progression. The main limitation of our study was the inclusion of only a single case with SNCA locus triplication. However, samples collected at two disease stages, shortly after PD onset and three years later, when dementia had developed, allowed longitudinal assessment. In conclusion, our findings provide new evidence for the pivotal role of the immune system in the development and progression of PD. Importantly, they suggest that PLTs may not only mirror central pathological processes but also actively participate in disease progression, and highlight the need to consider sex as a critical biological variable in PD research. Declarations Ethics approval and consent to participate: This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of the University Hospital Germans Trias i Pujol (Date February 25, 2022/No PI-22-024). Informed consent was obtained from all individual participants involved in the study. Competing Interests: The authors have no relevant financial or non-financial interests to disclose. Funding Declaration : This research was funded by Spain's Ministry of Science and Innovation, grant number PI21/00833, PI21/00886, PMP22/00100 and PI24/00214, integrated in the National R + D + I and funded by the ISCIII and the European Regional Development Fund. Author Contribution S.J. wrote the main text of the manuscript and prepared the figures and tables. S.J., J.M., D.A., A.T., and M.M. conducted the methodological aspects of the study, and A.M., D.S., L.I., D.V., R.A., and P.P. recruited the participants. K.B. and P.P. conceptualized the study, secured the funding, and reviewed the manuscript. All authors reviewed and approved the final manuscript. Acknowledgement The authors express their gratitude to all patients and volunteers for their cooperation and contributions. This research was funded by Spain's Ministry of Science and Innovation, grant number PI21/00833, PI21/00886, PMP22/00100 and PI24/00214, integrated in the National R + D + I and funded by the ISCIII and the European Regional Development Fund. Data Availability The data generated and analyzed during the current study are available from the corresponding author upon reasonable request. References Kalia, L. V.; Lang, A. E. Parkinson’s Disease. Lancet Lond. Engl. 2015, 386 (9996), 896–912. https://doi.org/10.1016/S0140-6736(14)61393-3 . Lindqvist, D.; Kaufman, E.; Brundin, L.; Hall, S.; Surova, Y.; Hansson, O. Non-Motor Symptoms in Patients with Parkinson’s Disease - Correlations with Inflammatory Cytokines in Serum. PloS One 2012, 7 (10), e47387. https://doi.org/10.1371/journal.pone.0047387 . Leiter, O.; Walker, T. L. Platelets in Neurodegenerative Conditions-Friend or Foe? Front. Immunol. 2020, 11 , 747. https://doi.org/10.3389/fimmu.2020.00747 . Beura, S. K.; Panigrahi, A. 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03:59:22","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":44041,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8422817/v1/7308aa33c01a1a5212f5af16.png"},{"id":100747982,"identity":"ec6ea704-494e-4cd8-be5f-da0a7d42984c","added_by":"auto","created_at":"2026-01-21 04:00:04","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":41126,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8422817/v1/a619fc89ef885c3219a09ea0.png"},{"id":100748024,"identity":"ab4a6d9c-39db-4e35-8430-9b563dcc3bed","added_by":"auto","created_at":"2026-01-21 04:00:17","extension":"xml","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":162440,"visible":true,"origin":"","legend":"","description":"","filename":"69c6e43a66c1436ba5b35dff2262c2771structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8422817/v1/f814e5aaeb77cd9cc64c0547.xml"},{"id":100747970,"identity":"9f975a91-7607-40ee-8b1f-a288ae958fdc","added_by":"auto","created_at":"2026-01-21 03:59:53","extension":"html","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":184994,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8422817/v1/b33c86bf51df937fb26e9a3e.html"},{"id":100748013,"identity":"f122946f-2fa2-4e95-ae60-99077e2d7d64","added_by":"auto","created_at":"2026-01-21 04:00:14","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":155948,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferentially expressed genes in PD vs CTRLs in blood and PLTs. \u003c/strong\u003eParkinson’s disease (PD), controls (CTRL), female PD (fPD), male PD (mPD), female controls (fCTRL) and male controls (mCTRL) (*p\u0026lt;0.05, **p\u0026lt;0.01).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8422817/v1/748e5cd69fcfeb6b9694068b.jpeg"},{"id":100747864,"identity":"89c17f41-f708-46ec-9204-619be3c68860","added_by":"auto","created_at":"2026-01-21 03:59:31","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":170929,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSNCA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003etranscript expression in blood and PLTs from patient SNCA3x. \u003c/strong\u003eEarly-stage PD (EPD), advanced PD with dementia (PDD), female controls (fCTRL).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8422817/v1/79971829caba564e25c35523.jpeg"},{"id":100748065,"identity":"70d2dc7e-ba40-4ae1-9432-7fe6ff486e01","added_by":"auto","created_at":"2026-01-21 04:00:33","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":123285,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression changes of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSNCA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e locus genes in blood and PLTs of patient SNCA3x\u003c/strong\u003e. Early-stage PD (EPD), advanced PD with dementia (PDD), female controls (fCTRL).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8422817/v1/8af42bd02c25b095b0ca2485.jpeg"},{"id":100747966,"identity":"df51a33d-cd78-4163-af9d-d417c474b57a","added_by":"auto","created_at":"2026-01-21 03:59:50","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":126830,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of 14-3-3 genes in PLTs of patient SNCA3x\u003c/strong\u003e. Early-stage PD (EPD), advanced PD with dementia (PDD), female controls (fCTRL).\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8422817/v1/8f6a3e61590a6f30b33eba47.jpeg"},{"id":100804085,"identity":"22229efe-d7eb-463e-8615-a9dfbc621e35","added_by":"auto","created_at":"2026-01-21 14:36:35","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":124770,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeatmaps summarizing PLT and blood expression changes characterizing PD progression in patient SNCA3x\u003c/strong\u003e. Early-stage PD (EPD), advanced PD with dementia (PDD).\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8422817/v1/4d4ace0193471484b974e283.jpeg"},{"id":102749013,"identity":"e2483c99-0b5d-4296-8eb0-fd4171d19cec","added_by":"auto","created_at":"2026-02-16 09:11:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1973300,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8422817/v1/8890aa34-d400-4649-b813-7245f9ff2e67.pdf"},{"id":100747788,"identity":"12b49cba-d386-49e0-a3c2-5f4bc23edd08","added_by":"auto","created_at":"2026-01-21 03:59:20","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":487321,"visible":true,"origin":"","legend":"","description":"","filename":"0Supplmaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8422817/v1/5bcf1ac278089441c8015210.docx"},{"id":100747874,"identity":"3a4370d5-9afc-46a6-9a7c-fe4d0878f5e1","added_by":"auto","created_at":"2026-01-21 03:59:37","extension":"jpeg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":122321,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8422817/v1/957ccfc10940b59d76117757.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"SNCA locus Triplication Drives Severe Parkinson’s Disease: Platelet and Blood Expression Profiles Evidence Systemic Involvement","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eParkinson disease (PD) is one of the most common neurodegenerative movement disorders, pathologically defined by abnormal aggregation of alpha-synuclein (AS), progressive loss of dopaminergic neurons in the substantia nigra, and chronic neuroinflammation\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Although its exact underlying mechanisms remain incompletely understood, aberrant protein homeostasis, oxidative stress, mitochondrial dysfunction, DNA damage, impaired neurotrophic signaling, and sustained inflammatory responses have all been implicated in disease pathogenesis. Increasing evidence now positions PD as a multisystem disorder characterized by widespread immune dysregulation and systemic inflammation\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePeripheral inflammatory mediators such as platelets (PLTs), have emerged as a successful model system for studying PD pathoprogression\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. PLTs share notable similarities with neurons in their degranulation/secretion patterns, vesicular trafficking, and surface receptor expression\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. They also store and release neurotransmitters and several neuron-specific proteins, including amyloid precursor protein (APP), β-amyloid (Aβ), tau protein, and brain-derived neurotrophic factor (BDNF)\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Remarkably, PLTs contain the highest concentration of AS per milligram of cellular protein in the blood\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e and express several proteins related to PD\u003csup\u003e7\u0026ndash;9\u003c/sup\u003e. Collectively, these features position PLTs as a suitable peripheral model for PD research.\u003c/p\u003e \u003cp\u003eDespite this, the role of peripheral inflammation in the progression of PD clinical symptoms remains unclear. Increasing evidence supports a multifactorial origin of PD, with the immune system positioned at the crossroads of genetic and environmental interactions that influence disease risk\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Although most cases are classified as sporadic with an unknown etiology, several risk factors \u0026ndash; including genetic predisposition \u0026ndash; have been proposed\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In this regard, point mutations, as well as duplications and triplications of the alpha-synuclein (\u003cem\u003eSNCA\u003c/em\u003e) gene, cause autosomal dominant familial PD. Affected carriers show clinical and pathological features of PD, and often PD with dementia (PDD) or dementia with Lewy bodies (DLB). However, whereas \u003cem\u003eSNCA\u003c/em\u003e duplications can be non-fully penetrant, \u003cem\u003eSNCA\u003c/em\u003e triplications are invariably associated with a rapidly progressive and aggressive PD phenotype\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. These observations demonstrate that both toxic gain-of-function from mutant protein and overproduction of wild-type protein promote AS aggregate formation\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo date, only six families with \u003cem\u003eSNCA\u003c/em\u003e triplication\u003csup\u003e\u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e and two apparently isolated patients\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e have been reported. The present case represents the ninth patient worldwide with \u003cem\u003eSNCA\u003c/em\u003e locus triplication, and the third reported isolated case.\u003c/p\u003e \u003cp\u003ePrevious triplication cases have described lengths of 0.351 Mb\u003csup\u003e20\u003c/sup\u003e, 1.2 Mb\u003csup\u003e15\u003c/sup\u003e and 2.61\u0026ndash;2.64 Mb\u003csup\u003e18\u003c/sup\u003e. A meta-analysis, including unpublished triplication cases, reported that the size of the \u003cem\u003eSNCA\u003c/em\u003e locus multiplication, both triplications and homozygous duplication, ranges between 2.14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.83 Mb\u003csup\u003e13\u003c/sup\u003e. Here, we aimed to investigate the molecular mechanisms underlying the onset of PD and its rapid progression to PD with dementia, based on a novel case of \u003cem\u003eSNCA\u003c/em\u003e triplication (SNCA3x). Using whole blood and PLT samples, we assessed whether immune- and inflammation-related pathways change during disease progression. In addition, we examined whether PLTs reflect molecular alterations typically observed in the brains of patients with PD. Therefore, we analyzed gene expression levels of genes located within the triplicated locus on chromosome 4q21.1, genes involved in inflammatory pathways, genes encoding 14-3-3 proteins, ribosomal genes, and the vesicular monoamine transporter gene VMAT2 (\u003cem\u003eSLC18A2\u003c/em\u003e) in both blood and PLTs.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eParticipants\u003c/h2\u003e \u003cp\u003eA patient carrying an \u003cem\u003eSNCA\u003c/em\u003e locus triplication (SNCA3x), with blood samples collected at two time points during the disease, was included. The first sample was obtained at age 41, corresponding to \u003cem\u003ede novo\u003c/em\u003e early-stage PD (EPD), and the second, at 44 years, when the patient presented advanced PD with dementia (PDD), after 3 years of disease duration. For comparison purposes, two additional cohorts were included. First, 15 age- and sex-matched early-onset PD patients, diagnosed according to the MDS clinical diagnostic criteria for Parkinson\u0026rsquo;s disease\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, were prospectively recruited during consultations at the Hospital Universitari Germans Trias i Pujol (Badalona, Spain). Second, 18 age- and sex-matched volunteers (CTRLs) without a family history of neurodegenerative disease were recruited as healthy controls at the Research Institute Germans Trias i Pujol (Badalona, Spain). The study was approved by the Clinical Research Ethics Committee of the University Hospital Germans Trias i Pujol (Date: February 25, 2022; No. PI-22-024), and all participants provided informed consent in accordance with the Declaration of Helsinki.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGenetic testing and copy number analyses\u003c/h3\u003e\n\u003cp\u003eGenomic DNA was purified from whole blood using standard methods. Genetic testing was performed using an in-house diagnostic panel for parkinsonism and dystonia (PD_DYT_2022_v2, IAD223176, and spike in IAD223184; Thermo Fisher Scientific, Waltham, MA, USA) and targeted NGS gene sequencing (Ion GeneStudio S5 System; Thermo Fisher Scientific). The genes included in the panel are shown in \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eMultiplex ligation-dependent probe amplification (MLPA) was performed to detect copy-number variations in PD-associated genes (PARK2, SNCA, PINK1, PARK7, ATP13A2) using the SALSA MLPA Parkinson Probemix 1 (MRC Holland, Amsterdam, the Netherlands), according to the manufacturer's instructions.\u003c/p\u003e \u003cp\u003eThe size of the triplicated region on chromosome 4q22.1 was determined by the CytoScan High Density Array (CytoScan HD Array, Applied Biosystems, Thermo Fisher Scientific), containing more than 2.6\u0026nbsp;million copy number markers and was processed on the GeneChip System 3000. Data was analyzed with the Chromosome Analysis Suite (ChAS) software.\u003c/p\u003e\n\u003ch3\u003eGene selection for expression analyses\u003c/h3\u003e\n\u003cp\u003eAmong the genes located within the triplicated \u003cem\u003eSNCA\u003c/em\u003e locus, seven genes (\u003cem\u003eSPP1\u003c/em\u003e, \u003cem\u003eABCG2\u003c/em\u003e, \u003cem\u003ePPM1K\u003c/em\u003e, \u003cem\u003eHERC6\u003c/em\u003e, \u003cem\u003eGPRIN3\u003c/em\u003e, \u003cem\u003eSNCA\u003c/em\u003e, \u003cem\u003eMMRN1\u003c/em\u003e) with relevance to neurodegenerative processes were selected. Additionally, eleven genes with established links to PD neuropathogenesis were also included: inflammatory pathway genes (\u003cem\u003eNLRP3\u003c/em\u003e, \u003cem\u003eTLR2\u003c/em\u003e, \u003cem\u003eTLR4\u003c/em\u003e); 14-3-3 protein\u0026ndash;encoding genes (\u003cem\u003eYWHAQ\u003c/em\u003e, \u003cem\u003eYWHAZ\u003c/em\u003e, \u003cem\u003eYWHAB\u003c/em\u003e, \u003cem\u003eYWHAG\u003c/em\u003e, \u003cem\u003eYWHAH\u003c/em\u003e); ribosomal genes (\u003cem\u003eRPS18\u003c/em\u003e, \u003cem\u003eRPL11\u003c/em\u003e); and the vesicular monoamine transporter gene \u003cem\u003eSLC18A2\u003c/em\u003e (VMAT2).\u003c/p\u003e\n\u003ch3\u003eRNA isolation and reverse transcription\u003c/h3\u003e\n\u003cp\u003eRNA from whole blood was extracted using the PAXGene Blood RNA Kit (PreAnalytiX, Hombrechtikon, Switzerland; Cat. no. 763134), and RNA from PLTs was extracted using the mirVANA miRNA Isolation Kit (Thermo Fisher Scientific; Cat. no. AM1561), following the manufacturer\u0026rsquo;s protocols. RNA concentration and purity were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific), with A260/A280 ratios adjusted to 1.8\u0026ndash;2.0, noting that PLTs-derived RNA may yield slightly lower values. Reverse transcription was performed using 1 \u0026micro;g of total RNA from blood and 200 ng from PLTs with Ready-to-go\u0026trade; You-Prime First-Strand Beads (Cytiva, Marlborough, MA, USA; Cat. no. 2796401).\u003c/p\u003e\n\u003ch3\u003ePrimer design and real-time PCR\u003c/h3\u003e\n\u003cp\u003eTranscript variant (tv)-specific forward primers targeted \u003cem\u003eSNCA\u003c/em\u003e exons 2a, 2b, or 1 for \u003cem\u003eSNCA\u003c/em\u003etv1, \u003cem\u003eSNCA\u003c/em\u003etv2, and \u003cem\u003eSNCA\u003c/em\u003etv3, respectively, with a common reverse primer in exon 4. Primer sequences and combinations are provided in \u003cb\u003eTable S2\u003c/b\u003e. Relative gene expression was assessed in blood and PLT samples by qPCR (LightCycler 480 I; Roche, Penzberg, Germany) using LUNA Universal qPCR Master Mix (New England Biolabs, Ipswich, MA, USA) in 14 \u0026micro;L reactions containing 1 \u0026micro;L cDNA. Reference genes were \u003cem\u003ePBGD1\u003c/em\u003e and \u003cem\u003eACTB\u003c/em\u003e for blood, and \u003cem\u003eRDX\u003c/em\u003e and \u003cem\u003eSNRDP3\u003c/em\u003e for PLTs\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. PD and CTRL samples were run in duplicate; samples corresponding to the two extractions from SNCA3x were run in quadruplicate. Standard curves for target and reference genes were generated in each run using serial dilutions of a control cDNA sample. Relative expression was calculated using the geometric averaging of multiple internal control genes\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAnalyses were performed using RStudio (4.3.2) and BioRender Graph software. Relative gene expression was calculated using the control group as the baseline for PD and the EPD group as the baseline for PDD. Data are presented as means. Normality and variance homogeneity were assessed using the Shapiro\u0026ndash;Wilk and Levene\u0026rsquo;s tests, respectively. For normally distributed data with equal variances, a two-tailed Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was applied; for unequal variances, a two-tailed Welch\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was used. For non-normally distributed data, the Mann\u0026ndash;Whitney \u003cem\u003eU\u003c/em\u003e test was applied. All p-values were adjusted for multiple testing using the FDR correction. Statistical significance was assessed based on FDR-adjusted p-values\u0026thinsp;\u0026le;\u0026thinsp;0.05. The two SNCA3x time points represent repeated measures in the same individual and were not subjected to inferential testing.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eClinical description of the SNCA3x carrier\u003c/h2\u003e \u003cp\u003eA female patient showed depressive symptoms and generalized bradykinesia at age 41, with a sudden onset of symptoms after the second in vitro fertilization treatment.\u003c/p\u003e \u003cp\u003eThe initial neurological examination revealed rigidity and altered tapering in upper extremities, slow alternating movements and a gait characterized by small steps, with her right leg slightly dragging. The UPDRS III score was 32, and the Hoehn and Yahr (HY) stage was 2. Dopamine transporter imaging (SPECT) revealed severe presynaptic dopaminergic depletion, and 123I-MIBG myocardial scintigraphy showed absent myocardial uptake, indicating postganglionic cardiac depletion. The patient was diagnosed with PD. After six months of treatment with levodopa, her symptoms improved moderately. With regards to her familial history, two paternal uncles developed parkinsonism at the ages of 55 and 75.\u003c/p\u003e \u003cp\u003eFifteen months after disease onset, she had memory complaints, and complained about more clumsiness of her right leg, dyskinesias, sialorrhea and wearing-off phenomenon. A neuropsychological battery revealed moderate to severe alteration of verbal and working memory, a marked slowing and increased response latency of spontaneous language.\u003c/p\u003e \u003cp\u003eEighteen months after disease onset, the patient experienced major functional limitation with the wearing-off phenomenon and significant nocturnal akinesia. Three months later, she complained of disturbing dyskinesias, especially in her right leg, and the wearing-off phenomenon predominantly in the morning. She developed nocturnal episodes compatible with REM sleep behavior disorder (RBD), and clonazepam 0,5 mg once a day was started.\u003c/p\u003e \u003cp\u003eThree years after the disease onset, she was on treatment with levodopa/benserazide 200 mg tid and amantadine 100 mg tid. A follow-up neuropsychological battery showed worse scores in response speed of spontaneous language, verbal and working memory, attention, executive functions, object naming and visuospatial functions.\u003c/p\u003e \u003cp\u003eIn her last neurological assessment, 5 years after disease onset, the UPDRS III score was 23, HY stage 2, and she was under levodopa/benserazide 200 mg tid, amantadine 100 mg tid, clonazepam 0,5 mg od, entacapone 200 mg qid. She was relatively autonomous but needed some aid when walking in the street.\u003c/p\u003e \u003cp\u003eDemographic data of PD patients and control individuals are shown in \u003cb\u003eTable S3\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGenetic testing\u003c/h2\u003e \u003cp\u003eNeither known disease-causing variants nor other rare variants were found in the 92 genes tested by targeted NGS. MLPA analysis revealed a double dose of the \u003cem\u003eSNCA\u003c/em\u003e gene, indicating the presence of either a homozygous duplication or a heterozygous triplication. To determine its origin, we analyzed samples from both parents, but neither of them carried the triplication. Thus, the patient harbored a \u003cem\u003ede novo SNCA\u003c/em\u003e triplication.\u003c/p\u003e \u003cp\u003eThe results of the CytoScan HD Array revealed that the triplication spans 2.41 Mb and encompasses 21 genes, 19 of which are protein-coding (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eGene expression differences in blood and PLTs are sex-dependent\u003c/h2\u003e \u003cp\u003eTo understand whether gene expression in whole blood and PLTs differed between PD and CTRLs, sex was included as a covariate. In blood, \u003cem\u003eSPP1\u003c/em\u003etv1, \u003cem\u003eSNCA\u003c/em\u003etv1 and \u003cem\u003eSNCA\u003c/em\u003etv2 were upregulated in PD vs. CTRLs (\u003cem\u003eSPP1\u003c/em\u003etv1: p\u0026thinsp;=\u0026thinsp;0.0064; \u003cem\u003eSNCA\u003c/em\u003etv1: p\u0026thinsp;=\u0026thinsp;0.0204; \u003cem\u003eSNCA\u003c/em\u003etv2: p\u0026thinsp;=\u0026thinsp;0.0237) (\u003cb\u003eFig.\u0026nbsp;1a-c\u003c/b\u003e). After sex-adjusted analysis, no differences were found among females, but all three transcripts remained significantly upregulated in male PD (mPD) vs. male CTRLs (mCTRLs) (\u003cem\u003eSPP1\u003c/em\u003etv1: p\u0026thinsp;=\u0026thinsp;0.00645; \u003cem\u003eSNCA\u003c/em\u003etv1: p\u0026thinsp;=\u0026thinsp;0.0204; \u003cem\u003eSNCA\u003c/em\u003etv2: p\u0026thinsp;=\u0026thinsp;0.0237) (\u003cb\u003eFig. S2a-c\u003c/b\u003e). Additionally, \u003cem\u003eSPP1\u003c/em\u003etv1 expression was increased in female CTRLs (fCTRLs) vs. mCTRLs (p\u0026thinsp;=\u0026thinsp;0.0397) (\u003cb\u003eFig.\u0026nbsp;1d\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eIn PLTs, \u003cem\u003eSNCA\u003c/em\u003etv2 and \u003cem\u003eSNCA\u003c/em\u003etv3 were downregulated (\u003cem\u003eSNCA\u003c/em\u003etv2: p\u0026thinsp;=\u0026thinsp;0.0325; \u003cem\u003eSNCA\u003c/em\u003etv3: p\u0026thinsp;=\u0026thinsp;0.027), and \u003cem\u003eMMRN1\u003c/em\u003e was upregulated (p\u0026thinsp;=\u0026thinsp;0.0464) in PD (\u003cb\u003eFig.\u0026nbsp;1e-g\u003c/b\u003e), but these changes lost significance after sex stratification (adjusted p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). \u003cem\u003eTLR2\u003c/em\u003e and \u003cem\u003eYWHAB\u003c/em\u003e were also downregulated in PD vs. CTRLs (\u003cb\u003eFig. S2d-e\u003c/b\u003e). When considering sex as a covariable, \u003cem\u003eTLR2\u003c/em\u003e, \u003cem\u003eTLR4\u003c/em\u003e and \u003cem\u003eYWHAB\u003c/em\u003e showed significantly decreased expression in female PD (fPD) compared to fCTRLs (\u003cem\u003eTLR2\u003c/em\u003e: p\u0026thinsp;=\u0026thinsp;0.0382; \u003cem\u003eTLR4\u003c/em\u003e: p\u0026thinsp;=\u0026thinsp;0.0187; \u003cem\u003eYWHAB\u003c/em\u003e: p\u0026thinsp;=\u0026thinsp;0.0343) (\u003cb\u003eFig.\u0026nbsp;1h-j\u003c/b\u003e), whereas only \u003cem\u003eYWHAB\u003c/em\u003e was decreased in mPD vs. mCTRLs (p\u0026thinsp;=\u0026thinsp;0.0349) (\u003cb\u003eFig. S2f\u003c/b\u003e). In addition, \u003cem\u003eTLR4\u003c/em\u003e was upregulated in mPD compared with fPD (p\u0026thinsp;=\u0026thinsp;0.035) (\u003cb\u003eFig.\u0026nbsp;1k\u003c/b\u003e). Upregulation of ribosomal genes \u003cem\u003eRPS18\u003c/em\u003e and \u003cem\u003eRPL11\u003c/em\u003e was observed in PD vs. CTRLs (\u003cem\u003eRPS18\u003c/em\u003e: p\u0026thinsp;=\u0026thinsp;0.0057; \u003cem\u003eRPL11\u003c/em\u003e: p\u0026thinsp;=\u0026thinsp;0.0015) (\u003cb\u003eFig.\u0026nbsp;2l-m\u003c/b\u003e); although, these differences remained statistically significant only in men (\u003cem\u003eRPS18\u003c/em\u003e: p\u0026thinsp;=\u0026thinsp;0.0219; \u003cem\u003eRPL11\u003c/em\u003e: p\u0026thinsp;=\u0026thinsp;0.0316) (\u003cb\u003eFig. S2g-h\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eDue to the strong sex-specific effects, and the fact that the SNCA3x patient was female, subsequent comparisons between early-stage PD (EPD) and PD dementia (PDD) were performed only with fPD and fCTRL cases.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 1. Differentially expressed genes in PD vs CTRLs in blood and PLTs.\u003c/b\u003e Parkinson\u0026rsquo;s disease (PD), controls (CTRL), female PD (fPD), male PD (mPD), female controls (fCTRL) and male controls (mCTRL) (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSNCA\u003c/b\u003e \u003cb\u003etranscript expression shifts depending on PD stage\u003c/b\u003e\u003c/p\u003e \u003cp\u003e Our initial results revealed marked divergence in \u003cem\u003eSNCA\u003c/em\u003e transcript variant expression. In blood, \u003cem\u003eSNCA\u003c/em\u003etv3 was upregulated by 27% in PDD compared with EPD, whereas \u003cem\u003eSNCA\u003c/em\u003etv1 and \u003cem\u003eSNCA\u003c/em\u003etv2 were downregulated (\u003cb\u003eFig.\u0026nbsp;2a\u003c/b\u003e). \u003cem\u003eSNCA\u003c/em\u003etv3 expression was 41% higher in EPD and 64% in PDD than fCTRLs (\u003cb\u003eFig.\u0026nbsp;2b\u003c/b\u003e). By contrast, in PLTs \u003cem\u003eSNCA\u003c/em\u003e transcript expression 214% higher in PDD than in EPD (\u003cb\u003eFig.\u0026nbsp;2c\u003c/b\u003e). Furthermore, PLT expression of \u003cem\u003eSNCA\u003c/em\u003etv1 was 32% lower in EPD and 77% higher in PDD, both compared with fCTRLs (\u003cb\u003eFig.\u0026nbsp;2d\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 2.\u003c/b\u003e \u003cb\u003eSNCA\u003c/b\u003e \u003cb\u003etranscript expression in blood and PLTs from patient SNCA3x.\u003c/b\u003e Early-stage PD (EPD), advanced PD with dementia (PDD), female controls (fCTRL).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSPP1\u003c/b\u003e \u003cb\u003etv1 is selectively overexpressed in blood during EPD\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eSPP1\u003c/em\u003etv1 expression in blood was approximately 50% higher than the other SNCA-locus genes in EPD and 56% higher in EPD than in PDD (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). In contrast, its expression was undetectable in PLTs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn EPD\u003c/b\u003e, \u003cb\u003eMMRN1\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ePPM1K\u003c/b\u003e \u003cb\u003eexpression changes are greater in PLTs than in blood\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn blood, the expression of the analyzed \u003cem\u003eSNCA\u003c/em\u003e locus genes was reduced by more than 40% in PDD (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), except for \u003cem\u003eMMRN1\u003c/em\u003e, whose expression was 50% higher than in EPD (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). No statistical differences were observed between fCTRLs and EPD. However, \u003cem\u003eMMRN1\u003c/em\u003e expression levels were 40% higher in PDD than in fCTRLs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). In PLTs, these differences were even more pronounced with an \u003cem\u003eMMRN1\u003c/em\u003e expression increase of 180% in PDD vs. EPD (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). In addition, its expression was 30% higher in EPD than in fCTRLs, and this difference increased to more than 100% in PDD (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). PLT \u003cem\u003ePPM1K\u003c/em\u003e expression in EPD was 150% higher than in fCTRLs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ef), 83% higher than in PDD and markedly elevated compared with other genes within the \u003cem\u003eSNCA\u003c/em\u003e locus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). By contrast, \u003cem\u003ePPM1K\u003c/em\u003e levels in EPD were comparable to those of other \u003cem\u003eSNCA\u003c/em\u003e-locus genes in whole blood (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePLT\u003c/b\u003e \u003cb\u003eSLC18A2\u003c/b\u003e \u003cb\u003edownregulation and upregulation of ribosomal genes\u003c/b\u003e \u003cb\u003eRPS18\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eRPL11\u003c/b\u003e \u003cb\u003echaracterize EPD\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSince PLTs and neurons share vesicle storage features, we analyzed \u003cem\u003eSLC18A2\u003c/em\u003e (VMAT2) expression and the ribosomal genes \u003cem\u003eRPS18\u003c/em\u003e and \u003cem\u003eRPL11\u003c/em\u003e to further assess PLT status. In whole blood, expression levels were too low to be quantified. In PLTs, however, \u003cem\u003eSLC18A2\u003c/em\u003e expression was markedly reduced in EPD, 250% lower than in PDD (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) and 104% lower than in fCTRLs. At the same time, no differences were observed between PDD and fCTRLs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). In contrast, \u003cem\u003eRPS18\u003c/em\u003e and \u003cem\u003eRPL11\u003c/em\u003e were upregulated in EPD by an average of 83% vs. PDD (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) and 150% vs. fCTRLs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003cb\u003e14-3-3 gene expression in PLTs is decreased in EPD\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGiven the modulatory role of 14-3-3 proteins in immune responses, we quantified PLT expression of \u003cem\u003eYWHAQ\u003c/em\u003e, \u003cem\u003eYWHAG\u003c/em\u003e, \u003cem\u003eYWHAZ\u003c/em\u003e, \u003cem\u003eYWHAH\u003c/em\u003e, and \u003cem\u003eYWHAB\u003c/em\u003e. Across disease stages, expression of 14-3-3\u0026ndash;encoding genes was on average 300% lower in EPD compared with PDD, with the largest difference observed for \u003cem\u003eYWHAB\u003c/em\u003e (750% lower in EPD) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). In EPD, \u003cem\u003eYWHAQ\u003c/em\u003e, \u003cem\u003eYWHAG\u003c/em\u003e, \u003cem\u003eYWHAZ\u003c/em\u003e, and \u003cem\u003eYWHAH\u003c/em\u003e were reduced on average by 75% compared with fCTRLs (\u003cb\u003eFig. S3a\u003c/b\u003e), while \u003cem\u003eYWHAB\u003c/em\u003e was decreased by 190% vs. fCTRLs and by 170% vs. fPD (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003e \u003cem\u003eYWHAQ\u003c/em\u003e, \u003cem\u003eYWHAG\u003c/em\u003e, and \u003cem\u003eYWHAZ\u003c/em\u003e were overexpressed by 24% in PDD compared with fCTRLs (\u003cb\u003eFig. S3b\u003c/b\u003e) and, in contrast, \u003cem\u003eYWHAB\u003c/em\u003e was 140% lower than in fCTRLs and 46% lower than in fPD (\u003cb\u003eFig. S3c\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eInflammatory genes are overexpressed early during PD progression\u003c/h2\u003e \u003cp\u003eTo assess potential differences in inflammatory pathways between EPD and PDD, we quantified PLT and blood expression of \u003cem\u003eTLR2\u003c/em\u003e, \u003cem\u003eTLR4\u003c/em\u003e, and \u003cem\u003eNLRP3\u003c/em\u003e. Overall, PLT inflammatory gene expression was 67% higher in EPD than in PDD (\u003cb\u003eFig. S4a\u003c/b\u003e). Compared with fCTRLs and fPD, EPD showed reduced \u003cem\u003eTLR2\u003c/em\u003e expression (44% and 77%, respectively), but an increased \u003cem\u003eTLR4\u003c/em\u003e expression (60% and 144%, respectively) (\u003cb\u003eFig. S4b\u003c/b\u003e). By contrast, \u003cem\u003eNLRP3\u003c/em\u003e expression was 63% lower in PDD than in fCTRLs, and both \u003cem\u003eTLR2\u003c/em\u003e and \u003cem\u003eTLR4\u003c/em\u003e were downregulated, by 154% and 125% vs. fCTRLs, and by 75% and 27% vs. fPD (\u003cb\u003eFig. S4c\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eIn blood, \u003cem\u003eTLR2\u003c/em\u003e, \u003cem\u003eTLR4\u003c/em\u003e, and \u003cem\u003eNLRP3\u003c/em\u003e expression was increased on average by 22% in PDD compared with EPD and reduced by 20% in EPD vs fCTRLs (\u003cb\u003eFig. S4d\u003c/b\u003e). In PDD, only a slight tendency to reduction was found compared with fCTRLs.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003e summarizes the results as heatmaps, highlighting both the impaired pathways and the magnitude of change. Expression shifts were far broader in PLTs (\u0026ndash;4.5 to 12.5) than in whole blood (\u0026ndash;0.02 to 1.2), indicating that PLTs offer a higher discriminatory potential for detecting expression differences.\u003c/p\u003e \u003cp\u003eProgression to advanced PD with dementia was associated with only modest expression shifts in blood, mainly a decrease in most \u003cem\u003eSNCA\u003c/em\u003e locus genes and an increased expression of \u003cem\u003eSNCA\u003c/em\u003etv3, \u003cem\u003eMMRN1\u003c/em\u003e, and inflammatory genes. By contrast, the PLT expression profile showed a pronounced deregulation. This included a moderate increase in inflammatory genes; marked upregulation of \u003cem\u003eSNCA\u003c/em\u003e transcripts, \u003cem\u003eMMRN1\u003c/em\u003e, 14-3-3 genes, and of the monoamine transporter VMAT2; as well as a moderate decrease in several \u003cem\u003eSNCA\u003c/em\u003e-locus-associated genes (\u003cem\u003eABCG2\u003c/em\u003e, \u003cem\u003ePPM1K\u003c/em\u003e, \u003cem\u003eHERC6\u003c/em\u003e, and \u003cem\u003eGPRIN3\u003c/em\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eWe report patient SNCA3x, presenting a rare, early-onset, rapidly progressing form of PD caused by a \u003cem\u003eSNCA\u003c/em\u003e locus triplication, which contains the largest number of protein-coding genes identified in an \u003cem\u003eSNCA\u003c/em\u003e locus triplication to date. Analysis of gene expression profile across EPD and PDD stages revealed that PLTs seem to capture pathogenic expression alterations, supporting their potential as peripheral biomarkers. The analysis of the expression profiles compared with a cohort of PD patients uncovered mechanisms shared with other PD forms, establishing the case SNCA3x as a powerful model for studying molecular processes relevant to disease progression.\u003c/p\u003e \u003cp\u003eGiven their modulatory role in immune responses and functional similarities to neurons, we explored whether PLTs may serve as indicators of expression changes associated with PD pathogenesis. Functional PLT alterations, including adhesion, activation, aggregation, and degranulation, have been reported in PD patients\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Multimerin-1, encoded by \u003cem\u003eMMRN1\u003c/em\u003e, which lies adjacent to \u003cem\u003eSNCA\u003c/em\u003e, is a PLT protein that mediates collagen-dependent adhesion. In our SNCA3x case, \u003cem\u003eMMRN1\u003c/em\u003e was included in the triplication, and its PLT and blood expression increased in a disease-stage-dependent manner, being higher in EPD than controls and highest in PDD. These results align with previous reports showing that \u003cem\u003eSNCA\u003c/em\u003e locus duplications or triplications encompassing the entire \u003cem\u003eMMRN1\u003c/em\u003e gene are associated with dementia, whereas those that do not fully include \u003cem\u003eMMRN1\u003c/em\u003e show no cognitive impairment\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Moreover, platelet \u003cem\u003eMMRN1\u003c/em\u003e expression was increased in patients with PD.\u003c/p\u003e \u003cp\u003eEmerging evidence increasingly supports a role of peripheral inflammation in the etiology of PD. A meta-analysis by Qu and colleagues\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e reported elevated levels of cytokines, including IL-6, TNF-α, CCL2, CX3CL1, and IL-β, in both blood and cerebrospinal fluid (CSF) of patients with PD, reinforcing peripheral inflammation as a hallmark of the disorder. In parallel, another study demonstrated that higher levels of systemic inflammatory markers were associated with reduced dopamine transporter (DAT) density in patients with PD\u003csup\u003e30\u003c/sup\u003e. Nevertheless, the extent to which peripheral inflammation contributes to PD phenotype progression remains unresolved, partly due to insufficient consideration of key modifiers, such as age of onset and sex. In our study, a significant reduction in PLT \u003cem\u003eTLR2\u003c/em\u003e and \u003cem\u003eTLR4\u003c/em\u003e expression was found in female PD patients compared with female CTRLs, and an upregulation of \u003cem\u003eTLR4\u003c/em\u003e in male PD patients compared with female PD patients.\u003c/p\u003e \u003cp\u003ePLT TLR activation is known to trigger pro-inflammatory signaling cascades\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, leading to α-granule release, and TLR2 in particular has been shown to induce PLT activation and aggregation\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Thus, reduced PLT TLR2 function may impair α-granule release, thereby limiting the secretion of MMRN1 and other signaling molecules. Moreover, a study by Koupenova and colleagues\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e reported higher expression levels of platelet TLR transcripts in women compared with men, suggesting that platelet \u003cem\u003eTLR\u003c/em\u003e expression is sex-dependent. Taken together, these findings indicate that in the early stages of PD, PLTs could not only lose part of their innate immune function but also exist in a state of pre-activation, in which α-granules accumulate rather than being secreted. Importantly, the observed sex-specific differences in PLT mRNA content, including higher \u003cem\u003eTLR4\u003c/em\u003e levels in men with PD, support the notion that women with PD may exhibit greater dysregulation of platelet \u003cem\u003eTLR\u003c/em\u003e expression. Nonetheless, further studies are required to delineate these sex-dependent differences and to determine their functional and clinical relevance.\u003c/p\u003e \u003cp\u003eIn addition to peripheral inflammation, our results revealed increased \u003cem\u003eSPP1\u003c/em\u003etv1 expression in whole blood from male PD patients compared with controls, and in EPD compared with PDD. SPP1 acts as a cytokine in inflammation, and previous studies in AD have reported elevated SPP1 in CSF and plasma\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. It has also been detected in plaque-associated microglia, where it has been proposed as a conserved disease-associated marker in both mice and humans\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Accordingly, our findings indicate elevated circulating SPP1 in PD, suggesting a novel source of this marker. These findings highlight the complexity of peripheral immune involvement and underscore the need for further analyses to elucidate not only their contribution to disease progression but also potential sex-specific differences.\u003c/p\u003e \u003cp\u003eAccumulating data also underscore the role of 14-3-3 proteins in modulating inflammatory responses\u003csup\u003e\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. In a previous study, we detected 14-3-3 expression changes in PLTs opposite to those observed in the temporal cortex, suggesting that PLTs may capture distinct molecular alterations that mirror and inversely reflect cerebral changes\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Furthermore, loss of 14-3-3 function has been linked to neurodegeneration in PD\u003csup\u003e41\u0026ndash;43\u003c/sup\u003e. Consistently, in PLTs, our results show a significant decrease in \u003cem\u003eYWHAB\u003c/em\u003e expression in PD and reduced 14-3-3 expression in EPD compared with PDD.\u003c/p\u003e \u003cp\u003eWe further observed reduced PLT monoamine transporter VMAT2 (\u003cem\u003eSLC18A2\u003c/em\u003e) levels in EPD, consistent with previous reports of decreased VMAT2 in PD PLTs\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. This observation aligns with evidence of impaired serotonin handling in PLT secretory vesicles (SVs) from patients with PD, characterized by reduced vesicular content, decreased uptake, diminished thrombin-induced release, and increased leakage\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. In the brain, VMAT2 is thought to play a protective role by limiting cytoplasmic dopamine accumulation, and thereby reducing ROS generation and oxidative stress\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. A similar mechanism may operate in PLTs, where reduced VMAT2 expression could foster an oxidative environment that increases cellular vulnerability. Supporting this view, we detected upregulation of \u003cem\u003ePPM1K\u003c/em\u003e in EPD. PPM1K, a mitochondrial matrix phosphatase essential for regulating the mitochondrial permeability transition pore\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, has been linked to mitochondrial dysfunction and neuronal loss when disrupted\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Notably, emerging evidence indicates functional crosstalk between peripheral tissues and the CNS in PD, with mitochondrial impairment as a potential common denominator\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Beyond mitochondrial alterations, PLT expression of the ribosomal genes \u003cem\u003eRPS18\u003c/em\u003e and \u003cem\u003eRPL11\u003c/em\u003e was increased in male PD patients compared with male CTRLs, and in EPD compared with PDD. Together, these findings point to a broader dysregulation of PLT molecular profiles not only across disease stages, but also in dependency on sex.\u003c/p\u003e \u003cp\u003eBuilding on these findings, we investigated \u003cem\u003eSNCA\u003c/em\u003e transcript-specific changes in blood and PLTs. While blood samples showed increased expression of \u003cem\u003eSNCA\u003c/em\u003etv1 and \u003cem\u003eSNCA\u003c/em\u003etv2 in PD, PLT expression revealed lower levels of \u003cem\u003eSNCA\u003c/em\u003etv2 and \u003cem\u003eSNCA\u003c/em\u003etv3. Moreover, between the EPD and PDD stages, transcript-specific changes diverged between blood and platelets, with all three transcripts showing increased PLT expression in PDD. Previous studies reported higher α-synuclein levels in blood from PD patients without dementia compared to those with dementia\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, consistent with our observations.\u003c/p\u003e \u003cp\u003eAlthough studies on synucleins in PLTs are scarce and sometimes inconsistent, our results position PLTs as a primary peripheral source for monitoring transcriptional changes associated with PD progression.\u003c/p\u003e \u003cp\u003eThe main limitation of our study was the inclusion of only a single case with \u003cem\u003eSNCA\u003c/em\u003e locus triplication. However, samples collected at two disease stages, shortly after PD onset and three years later, when dementia had developed, allowed longitudinal assessment.\u003c/p\u003e \u003cp\u003eIn conclusion, our findings provide new evidence for the pivotal role of the immune system in the development and progression of PD. Importantly, they suggest that PLTs may not only mirror central pathological processes but also actively participate in disease progression, and highlight the need to consider sex as a critical biological variable in PD research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics approval and consent to participate:\u003c/h2\u003e \u003cp\u003eThis study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of the University Hospital Germans Trias i Pujol (Date February 25, 2022/No PI-22-024).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eInformed consent\u003c/strong\u003e \u003cp\u003ewas obtained from all individual participants involved in the study.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting Interests:\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003e \u003cb\u003eDeclaration\u003c/b\u003e: This research was funded by Spain's Ministry of Science and Innovation, grant number PI21/00833, PI21/00886, PMP22/00100 and PI24/00214, integrated in the National R\u0026thinsp;+\u0026thinsp;D\u0026thinsp;+\u0026thinsp;I and funded by the ISCIII and the European Regional Development Fund.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eS.J. wrote the main text of the manuscript and prepared the figures and tables. S.J., J.M., D.A., A.T., and M.M. conducted the methodological aspects of the study, and A.M., D.S., L.I., D.V., R.A., and P.P. recruited the participants. K.B. and P.P. conceptualized the study, secured the funding, and reviewed the manuscript. All authors reviewed and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors express their gratitude to all patients and volunteers for their cooperation and contributions. This research was funded by Spain's Ministry of Science and Innovation, grant number PI21/00833, PI21/00886, PMP22/00100 and PI24/00214, integrated in the National R + D + I and funded by the ISCIII and the European Regional Development Fund.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data generated and analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKalia, L. V.; Lang, A. E. Parkinson\u0026rsquo;s Disease. \u003cem\u003eLancet Lond. 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Propagation of Alpha-Synuclein Pathology from the Olfactory Bulb: Possible Role in the Pathogenesis of Dementia with Lewy Bodies. \u003cem\u003eCell Tissue Res.\u003c/em\u003e 2018, \u003cem\u003e373\u003c/em\u003e (1), 233\u0026ndash;243. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00441-017-2733-6\u003c/span\u003e\u003cspan address=\"10.1007/s00441-017-2733-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Parkinson’s disease, SNCA locus triplication, gene expression, platelets, 14-3-3, inflammation, peripheral biomarker","lastPublishedDoi":"10.21203/rs.3.rs-8422817/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8422817/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eParkinson disease (PD) is increasingly recognized as a multisystem condition with substantial immune involvement. Functional parallels described between neurons and platelets have raised interest in the latter as a window into neurodegenerative disease mechanisms. While most PD cases are sporadic, rarely, mutations or gene dosage increases of \u003cem\u003eSNCA\u003c/em\u003e cause familial PD. We report the case of the ninth PD patient carrying a \u003cem\u003eSNCA\u003c/em\u003e locus triplication on chromosome 4q21.1 (SNCA3x), characterized by early-onset dementia and encompassing the largest number of coding genes described to date. To explore molecular correlates of disease progression, we longitudinally profiled the gene expression in blood and platelets from the SNCA3x PD patient in early and advanced stages, compared with healthy controls and early-onset PD patients stratified by age and sex. \u0026nbsp;Early-stage PD was characterized by the downregulation of 14-3-3 and monoamine transporter genes, along with the upregulation of ribosomal genes in platelets, \u003cem\u003eSPP1\u003c/em\u003etv1 in blood, and of inflammatory and PPM1K phosphatase genes in both blood and platelets. In the advanced stage of PD, we observed overexpression of \u003cem\u003eSNCA\u003c/em\u003e and \u003cem\u003eMMRN1\u003c/em\u003ein blood and platelets. These dynamic shifts suggest a stage-dependent platelet signature that mirrors neurodegenerative and immune pathways. Our findings reveal shared molecular mechanisms with other forms of PD and underscore platelets as a peripheral source of biomarker discovery in PD.\u003c/p\u003e","manuscriptTitle":"SNCA locus Triplication Drives Severe Parkinson’s Disease: Platelet and Blood Expression Profiles Evidence Systemic Involvement","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-21 03:57:45","doi":"10.21203/rs.3.rs-8422817/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"43f703dc-f389-494f-971f-0d3d19204ae8","owner":[],"postedDate":"January 21st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":61266682,"name":"Health sciences/Biomarkers"},{"id":61266683,"name":"Health sciences/Diseases"},{"id":61266684,"name":"Biological sciences/Genetics"},{"id":61266685,"name":"Health sciences/Neurology"},{"id":61266686,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2026-02-15T19:53:57+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-21 03:57:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8422817","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8422817","identity":"rs-8422817","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}