DECIPHERING THE GENETIC ARCHITECTURE OF PARKINSON’S DISEASE IN THE LUXEMBOURGISH POPULATION

Parkinson's Disease (PD) is the fastest-growing neurodegenerative disorder (Bloem et al., 2021; Dorsey et al., 2018). It is characterized by motor symptoms such as postural instability, rest tremor, bradykinesia and rigidity (Kalia and Lang, 2015). Additionally, PD exhibits non-motor symptoms including cognitive impairment, neuropsychiatric features like hallucinations, depression, and anxiety, as well as sleep disturbances, hyposmia and autonomic dysfunction (Khoo et al., 2013). While the exact causes of PD remain incompletely understood, research has identified genetic risk factors as important contributing factors in its development. Although PD appears typically as sporadic, approximately 30% of cases can be linked to genetic factors involving monogenic forms (Billingsley et al., 2018). Although motor symptoms can be effectively addressed by pharmacological therapies at least in early disease stages, there is currently no treatment that may interfere with the chronic progressive neurodegeneration. Therefore, there is a strong motivation among clinicians and researchers to perform early diagnoses and identify genetic variants that could lead to future causative and thereby neuroprotective therapies. However, PD is a complex disorder influenced by a combination of environmental and genetic factors. Only a limited subset of genes have been conclusively associated with typical PD, as their causative role in Mendelian forms of PD has been consistently replicated in multiple studies involving large populations of PD patients (Bandres-Ciga et al., 2020). These rare Mendelian forms follow distinct inheritance patterns and have a notable impact on disease development (e.g., SNCA, LRRK2, VPS35, PRKN, PINK1 and PARK7). There are also low-frequency variants with significant effects, such as the GBA1 and LRRK2 genes. These variants are not as rare as monogenic mutations, but their frequency remains relatively low in the general population. Then, there are the common variants that exert minor effects and highlight genetic variants that are prevalent in the population but individually contribute only a modest risk toward PD (Manolio et al., 2009). Although not as extensively studied as single nucleotide variants, small copy number variants have received considerable interest due to their potential pathogenic implications (La Cognata et al., 2017; Pankratz et al., 2011; Toft and Ross, 2010). As part of the Luxembourg Parkinson's study, which includes both healthy individuals and patients with PD and atypical parkinsonism, we aim to investigate the genetic background of PD-causal genes (LRRK2, SNCA, VPS35, PRKN, PARK7, PINK1 and ATP13A2) and other known PD-associated genes, looking for rare Single-Nucleotide Variants (SNVs), Copy Number Variation (CNVs), and estimating the effect of multiple common SNVs in predicting an individual's probability of developing PD, using Polygenic Risk Scores (PRS). The Luxembourg Parkinson's study, a large monocentric longitudinal cohort, included 1791 participants, 911 of whom were diagnosed cases and 880 neurologically healthy controls. The mean age of the control group was 64.7 ± 12.1 years in 2023. The mean age at onset for PD patients was 62.4 ± 11.7 years. We identified 12.1% of PD patients and 5% of healthy controls carried GBA1 variants. Additionally, four GBA1 variants were discovered in patients with progressive supranuclear palsy and dementia with Lewy bodies. We identified different categories of pathogenic GBA1 variants, including those with severe, mild and risk-associated effects. We then studied the relationships between genotypes and phenotypes, to better understand the impact of each type of variant and how they contribute to disease severity. We discovered a total of 60 rare SNVs within seven PD-causal genes. Notably, nine of these variants were found to be disease-causing in LRRK2, PINK1, and PRKN genes. Additionally, we identified eleven rare CNVs in the PRKN gene, encompassing seven duplications and four deletions. We showed that the PRSs were significantly associated with PD and highlighted the important role of polygenic background plays in modulating PD risk in carriers of pathogenic GBA1 variants. Moreover, in an explorative study, where we are looking for loss of function variants, we identified 134 rare variants and eight rare copy number variations in PD-related genes, that could potentially contribute to PD, but these results are not statistically reliable and further analysis is required. The failure to discover novel genetic variants with whole-genome sequencing data can be attributed to the limited power to detect rare variants with small effects, as well as the relatively small sample size of the study. We carried out an in-depth genetic analysis of the participants in the Luxembourg Parkinson's study. Our findings should help future research to unravel the complex genetic landscape of PD. This knowledge will make it possible to classify participants according to their genetic profile, improving the effectiveness of future precision medicine approaches. These targeted therapies can then be tailored to attack specific molecular targets, paving the way for a new era of personalized treatment strategies

Body-First Subtype of Parkinson's Disease with Probable REM-Sleep Behavior Disorder Is Associated with Non-Motor Dominant Phenotype

Background: The hypothesis of body-first vs. brain-first subtype of PD has been proposed with REM-Sleep behavior disorder (RBD) defining the former. The body-first PD presumes an involvement of the brainstem in the pathogenic process with higher burden of autonomic dysfunction. Objective: To identify distinctive clinical subtypes of idiopathic Parkinson’s disease (iPD) in line with the formerly proposed concept of body-first vs. brain-first subtypes in PD, we analyzed the presence of probable RBD (pRBD), sex, and the APOE ɛ4 carrier status as potential sub-group stratifiers. Methods: A total of 400 iPD patients were included in the cross-sectional analysis from the baseline dataset with a completed RBD Screening Questionnaire (RBDSQ) for classifying as pRBD by using the cut-off RBDSQ≥6. Multiple regression models were applied to explore (i) the effect of pRBD on clinical outcomes adjusted for disease duration and age, (ii) the effect of sex on pRBD, and (iii) the association of APOE ɛ4 and pRBD. Results: iPD-pRBD was significantly associated with autonomic dysfunction (SCOPA-AUT), level of depressive symptoms (BDI-I), MDS-UPDRS I, hallucinations, and constipation, whereas significantly negatively associated with quality of life (PDQ-39) and sleep (PDSS). No significant association between sex and pRBD or APOE ɛ4 and pRBD in iPD was found nor did we determine a significant effect of APOE ɛ4 on the PD phenotype. Conclusion: We identified an RBD-specific PD endophenotype, characterized by predominant autonomic dysfunction, hallucinations, and depression, corroborating the concept of a distinctive body-first subtype of PD. We did not observe a significant association between APOE ɛ4 and pRBD suggesting both factors having an independent effect on cognitive decline in iPD

Accurate long-read sequencing identified GBA1 as major risk factor in the Luxembourgish Parkinson's study.

peer reviewedHeterozygous variants in the glucocerebrosidase GBA1 gene are an increasingly recognized risk factor for Parkinson's disease (PD). Due to the GBAP1 pseudogene, which shares 96% sequence homology with the GBA1 coding region, accurate variant calling by array-based or short-read sequencing methods remains a major challenge in understanding the genetic landscape of GBA1-associated PD. We analyzed 660 patients with PD, 100 patients with Parkinsonism and 808 healthy controls from the Luxembourg Parkinson's study, sequenced using amplicon-based long-read DNA sequencing technology. We found that 12.1% (77/637) of PD patients carried GBA1 variants, with 10.5% (67/637) of them carrying known pathogenic variants (including severe, mild, risk variants). In comparison, 5% (34/675) of the healthy controls carried GBA1 variants, and among them, 4.3% (29/675) were identified as pathogenic variant carriers. We found four GBA1 variants in patients with atypical parkinsonism. Pathogenic GBA1 variants were 2.6-fold more frequently observed in PD patients compared to controls (OR = 2.6; CI = [1.6,4.1]). Three novel variants of unknown significance (VUS) were identified. Using a structure-based approach, we defined a potential risk prediction method for VUS. This study describes the full landscape of GBA1-related parkinsonism in Luxembourg, showing a high prevalence of GBA1 variants as the major genetic risk for PD. Although the long-read DNA sequencing technique used in our study may be limited in its effectiveness to detect potential structural variants, our approach provides an important advancement for highly accurate GBA1 variant calling, which is essential for providing access to emerging causative therapies for GBA1 carriers.R-AGR-0592 - FNR - NCER-PD Phase II Coordination (01/06/2015 - 30/11/2023) - KRÜGER Rejko3. Good health and well-bein

Single-cell sequencing of human midbrain reveals glial activation and a Parkinson-specific neuronal state.

Idiopathic Parkinson's disease is characterized by a progressive loss of dopaminergic neurons, but the exact disease etiology remains largely unknown. To date, Parkinson's disease research has mainly focused on nigral dopaminergic neurons, although recent studies suggest disease-related changes also in non-neuronal cells and in midbrain regions beyond the substantia nigra. While there is some evidence for glial involvement in Parkinson's disease, the molecular mechanisms remain poorly understood. The aim of this study was to characterize the contribution of all cell types of the midbrain to Parkinson's disease pathology by single-nuclei RNA sequencing and to assess the cell type-specific risk for Parkinson's disease employing the latest genome-wide association study. We profiled >41 000 single-nuclei transcriptomes of postmortem midbrain from six idiopathic Parkinson's disease patients and five age-/sex-matched controls. To validate our findings in a spatial context, we utilized immunolabeling of the same tissues. Moreover, we analyzed Parkinson's disease-associated risk enrichment in genes with cell type-specific expression patterns. We discovered a neuronal cell cluster characterized by CADPS2 overexpression and low TH levels, which was exclusively present in IPD midbrains. Validation analyses in laser-microdissected neurons suggest that this cluster represents dysfunctional dopaminergic neurons. With regard to glial cells, we observed an increase in nigral microglia in Parkinson's disease patients. Moreover, nigral idiopathic Parkinson's disease microglia were more amoeboid, indicating an activated state. We also discovered a reduction in idiopathic Parkinson's disease oligodendrocyte numbers with the remaining cells being characterized by a stress-induced upregulation of S100B. Parkinson's disease risk variants were associated with glia- and neuron-specific gene expression patterns in idiopathic Parkinson's disease cases. Furthermore, astrocytes and microglia presented idiopathic Parkinson's disease-specific cell proliferation and dysregulation of genes related to unfolded protein response and cytokine signaling. While reactive patient astrocytes showed CD44 overexpression, idiopathic Parkinson's disease-microglia revealed a pro-inflammatory trajectory characterized by elevated levels of IL1B, GPNMB, and HSP90AA1. Taken together, we generated the first single-nuclei RNA sequencing dataset from the idiopathic Parkinson's disease midbrain, which highlights a disease-specific neuronal cell cluster as well as 'pan-glial' activation as a central mechanism in the pathology of the movement disorder. This finding warrants further research into inflammatory signaling and immunomodulatory treatments in Parkinson's disease

Age at onset as stratifier in idiopathic Parkinson’s disease – effect of ageing and polygenic risk score on clinical phenotypes

Several phenotypic differences observed in Parkinson’s disease (PD) patients have been linked to age at onset (AAO). We endeavoured to find out whether these differences are due to the ageing process itself by using a combined dataset of idiopathic PD (n = 430) and healthy controls (HC; n = 556) excluding carriers of known PD-linked genetic mutations in both groups. We found several significant effects of AAO on motor and non-motor symptoms in PD, but when comparing the effects of age on these symptoms with HC (using age at assessment, AAA), only positive associations of AAA with burden of motor symptoms and cognitive impairment were significantly different between PD vs HC. Furthermore, we explored a potential effect of polygenic risk score (PRS) on clinical phenotype and identified a significant inverse correlation of AAO and PRS in PD. No significant association between PRS and severity of clinical symptoms was found. We conclude that the observed non-motor phenotypic differences in PD based on AAO are largely driven by the ageing process itself and not by a specific profile of neurodegeneration linked to AAO in the idiopathic PD patients

Genetic landscape of Parkinson’s disease and related diseases in Luxembourg

peer reviewedObjectives: To explore the genetic architecture of PD in the Luxembourg Parkinson’s Study including cohorts of healthy people and patients with Parkinson’s disease (PD) and atypical parkinsonism (AP).Methods: 809 healthy controls, 680 PD and 103 AP were genotyped using the Neurochip array. We screened and validated rare single nucleotide variants (SNVs) and copy number variants (CNVs) within seven PD-causing genes (LRRK2, SNCA, VPS35, PRKN, PARK7, PINK1 and ATP13A2). Polygenic risk scores (PRSs) were generated using the latest genome-wide association study for PD. We then estimated the role of common variants in PD risk by applying gene-set-specific PRSs.Results: We identified 60 rare SNVs in seven PD-causing genes, nine of which were pathogenic in LRRK2, PINK1 and PRKN. Eleven rare CNVs were detected in PRKN including seven duplications and four deletions. The majority of PRKN SNVs and CNVs carriers were heterozygous and not differentially distributed between cases and controls. The PRSs were significantly associated with PD and identified specific molecular pathways related to protein metabolism and signal transduction as drivers of PD risk.Conclusion: We performed a comprehensive genetic characterization of the deep-phenotyped individuals of the Luxembourgish Parkinson’s Study. Heterozygous SNVs and CNVs in PRKN were not associated with higher PD risk. In particular, we reported novel digenic variants in PD related genes and rare LRRK2 SNVs in AP patients. Our findings will help future studies to unravel the genetic complexity of PD.3. Good health and well-bein

Single-cell sequencing of the human midbrain reveals glial activation and a neuronal state specific to Parkinson’s disease

Parkinson’s disease (PD) etiology is associated with genetic and environmental factors that lead to a loss of dopaminergic neurons. However, the functional interpretation of PD-associated risk variants and how other midbrain cells contribute to this neurodegenerative process are poorly understood. Here, we profiled >41,000 single-nuclei transcriptomes of postmortem midbrain tissue from 6 idiopathic PD (IPD) patients and 5 matched controls. We show that PD-risk variants are associated with glia- and neuron-specific gene expression patterns. Furthermore, Microglia and astrocytes presented IPD-specific cell proliferation and dysregulation of genes related to unfolded protein response and cytokine signalling. IPD-microglia revealed a specific pro-inflammatory trajectory. Finally, we discovered a neuronal cell cluster exclusively present in IPD midbrains characterized by CADPS2 overexpression and a high proportion of cycling cells. We conclude that elevated CADPS2 expression is specific to dysfunctional dopaminergic neurons, which have lost their dopaminergic identity and unsuccessful attempt to re-enter the cell cycle

Accurate long-read sequencing identified GBA1 as major risk factor in the Luxembourgish Parkinson’s study

Heterozygous variants in the glucocerebrosidase GBA1 gene are an increasingly recognized risk factor for Parkinson’s disease (PD). Due to the GBAP1 pseudogene, which shares 96% sequence homology with the GBA1 coding region, accurate variant calling by array-based or short-read sequencing methods remains a major challenge in understanding the genetic landscape of GBA1-associated PD. We analyzed 660 patients with PD, 100 patients with Parkinsonism and 808 healthy controls from the Luxembourg Parkinson’s study, sequenced using amplicon-based long-read DNA sequencing technology. We found that 12.1% (77/637) of PD patients carried GBA1 variants, with 10.5% (67/637) of them carrying known pathogenic variants (including severe, mild, risk variants). In comparison, 5% (34/675) of the healthy controls carried GBA1 variants, and among them, 4.3% (29/675) were identified as pathogenic variant carriers. We found four GBA1 variants in patients with atypical parkinsonism. Pathogenic GBA1 variants were 2.6-fold more frequently observed in PD patients compared to controls (OR = 2.6; CI = [1.6,4.1]). Three novel variants of unknown significance (VUS) were identified. Using a structure-based approach, we defined a potential risk prediction method for VUS. This study describes the full landscape of GBA1-related parkinsonism in Luxembourg, showing a high prevalence of GBA1 variants as the major genetic risk for PD. Although the long-read DNA sequencing technique used in our study may be limited in its effectiveness to detect potential structural variants, our approach provides an important advancement for highly accurate GBA1 variant calling, which is essential for providing access to emerging causative therapies for GBA1 carriers