Use of sera cell free DNA (cfDNA) and exovesicle-DNA for the molecular diagnosis of chronic Chagas disease.

Chagas disease, a neglected tropical disease, is now considered a worldwide health concern as a result of migratory movements from Central and South America to other regions that were considered free of the disease, and where the epidemiological risk is limited to transplacental transmission or blood or organ donations from infected persons. Parasite detection in chronically ill patients is restricted to serological tests that only determine infection by previous infection and not the presence of the parasite, especially in patients undergoing treatment evaluation or in newborns. We have evaluated the use of nucleic acids from both circulating exovesicles and cell-free DNA (cfDNA) from 50 samples twice randomly selected from a total of 448 serum samples from immunologically diagnosed patients in whom the presence of the parasite was confirmed by nested PCR on amplicons resulting from amplification with kinetoplastid DNA-specific primers 121F-122R. Six samples were randomly selected to quantify the limit of detection by qPCR in serum exovesicles. When the nucleic acids thus purified were assayed as a template and amplified with kinetoplastid DNA and nuclear satellite DNA primers, a 100% positivity rate was obtained for all positive samples assayed with kDNA-specific primers and 96% when SAT primers were used. However, isolation of cfDNA for Trypanosoma cruzi and amplification with SAT also showed 100% positivity. The results demonstrate that serum exovesicles contain DNA of mitochondrial and nuclear origin, which can be considered a mixed population of exovesicles of parasitic origin. The results obtained with serum samples prove that both cfDNA and Exovesicle DNA can be used to confirm parasitaemia in chronically ill patients or in samples where it is necessary to demonstrate the active presence of the parasite. The results confirm for the first time the existence of exovesicles of mitochondrial origin of the parasite in the serum of those affected by Chagas disease.

Brainstem neuromelanin and iron MRI reveals a precise signature for idiopathic and LRRK2 Parkinson's disease.

Neuromelanin (NM) loss in substantia nigra pars compacta (SNc) and locus coeruleus (LC) reflects neuronal death in Parkinson's disease (PD). Since genetically-determined PD shows varied clinical expressivity, we wanted to accurately quantify and locate brainstem NM and iron, to discover whether specific MRI patterns are linked to Leucine-rich repeat kinase 2 G2019S PD (LRRK2-PD) or idiopathic Parkinson's disease (iPD). A 3D automated MRI atlas-based segmentation pipeline (3D-ABSP) for NM/iron-sensitive MRI images topographically characterized the SNc, LC, and red nucleus (RN) neuronal loss and calculated NM/iron contrast ratio (CR) and normalized volume (nVol). Left-side NM nVol was larger in all groups. PD had lower NM CR and nVol in ventral-caudal SNc, whereas iron increased in lateral, medial-rostral, and caudal SNc. The SNc NM CR reduction was associated with psychiatric symptoms. LC CR and nVol discriminated better among subgroups: LRRK2-PD had similar LC NM CR and nVol as that of controls, and larger LC NM nVol and RN iron CR than iPD. PD showed higher iron SNc nVol than controls, especially among LRRK2-PD. ROC analyses showed an AUC > 0.92 for most pairwise subgroup comparisons, with SNc NM being the best discriminator between HC and PD. NM measures maintained their discriminator power considering the subgroup of PD patients with less than 5 years of disease duration. The SNc iron CR and nVol increase was associated with longer disease duration in PD patients. The 3D-ABSP sensitively identified NM and iron MRI patterns strongly correlated with phenotypic PD features.

Implementation of Exome Sequencing in Clinical Practice for Neurological Disorders.

Neurological disorders (ND) are diseases that affect the brain and the central and autonomic nervous systems, such as neurodevelopmental disorders, cerebellar ataxias, Parkinson's disease, or epilepsies. Nowadays, recommendations of the American College of Medical Genetics and Genomics strongly recommend applying next generation sequencing (NGS) as a first-line test in patients with these disorders. Whole exome sequencing (WES) is widely regarded as the current technology of choice for diagnosing monogenic ND. The introduction of NGS allows for rapid and inexpensive large-scale genomic analysis and has led to enormous progress in deciphering monogenic forms of various genetic diseases. The simultaneous analysis of several potentially mutated genes improves the diagnostic process, making it faster and more efficient. The main aim of this report is to discuss the impact and advantages of the implementation of WES into the clinical diagnosis and management of ND. Therefore, we have performed a retrospective evaluation of WES application in 209 cases referred to the Department of Biochemistry and Molecular Genetics of the Hospital Clinic of Barcelona for WES sequencing derived from neurologists or clinical geneticists. In addition, we have further discussed some important facts regarding classification criteria for pathogenicity of rare variants, variants of unknown significance, deleterious variants, different clinical phenotypes, or frequency of actionable secondary findings. Different studies have shown that WES implementation establish diagnostic rate around 32% in ND and the continuous molecular diagnosis is essential to solve the remaining cases.

Elevated urine BMP phospholipids in LRRK2 and VPS35 mutation carriers with and without Parkinson's disease.

Elevated urine bis(monoacylglycerol)phosphate (BMP) levels have been found in gain-of-kinase function LRRK2 G2019S mutation carriers. Here, we have expanded urine BMP analysis to other Parkinson's disease (PD) associated mutations and found them to be consistently elevated in carriers of LRRK2 G2019S and R1441G/C as well as VPS35 D620N mutations. Urine BMP levels are promising biomarkers for patient stratification and potentially target engagement in clinical trials of emerging targeted PD therapies.

Differential serum microRNAs in premotor LRRK2 G2019S carriers from Parkinson's disease.

The LRRK2 G2019S pathogenic mutation causes LRRK2-associated Parkinson's disease (L2PD) with incomplete penetrance. LRRK2 non-manifesting carriers (L2NMC) are at PD high risk but predicting pheno-conversion is challenging given the lack of progression biomarkers. To investigate novel biomarkers for PD premotor stages, we performed a longitudinal microRNA (miRNA) assessment of serum samples from G2019S L2NMC followed-up over 8 years. Our cohort consisted of G2019S L2NMC stratified by dopamine transporter single-photon emission computed tomography (DaT-SPECT) into DaT-negative (n = 20) and DaT-positive L2NMC (n = 20), pheno-converted G2019S L2PD patients (n = 20), idiopathic PD (iPD) (n = 19), and controls (n = 40). We also screened a second cohort of L2PD patients (n = 19) and controls (n = 20) (Total n = 158). Compared to healthy controls, we identified eight deregulated miRNAs in DaT-negative L2NMC, six in DaT-positive L2NMC, and one in L2PD. Between groups, the highest miRNA differences, 24 candidate miRNAs, occurred between DaT-positive L2NMC and L2PD. Longitudinally, we found 11 common miRNAs with sustained variation in DaT-negative and DaT-positive L2NMCs compared to their baselines. Our study identifies novel miRNA alterations in premotor stages of PD co-occurring with progressive DaT-SPECT decline before motor manifestation, whose deregulation seems to attenuate after the diagnosis of L2PD. Moreover, we identified four miRNAs with relatively high discriminative ability (AUC = 0.82) between non-pheno-converted DaT-positive G2019S carriers and pheno-converted L2PD patients (miR-4505, miR-8069, miR-6125, and miR-451a), which hold potential as early progression biomarkers for PD.

Cell-free mitochondrial DNA deletions in idiopathic, but not LRRK2, Parkinson's disease.

Mitochondrial dysfunction happens in both idiopathic (iPD) and LRRK2-related Parkinson's disease (LRRK2-PD). Nonetheless, previous studies suggested that a different type of mitochondrial pathology underlies the neurodegeneration in these two disorders. To further explore this hypothesis, we developed a novel multiplex digital PCR assay that allows the absolute quantification of cell-free mitochondrial DNA (cf-mtDNA) copy number and deletion ratio directly in cerebrospinal fluid (CSF) by simultaneously measuring two opposed regions of the mtDNA circular molecule, one of them in the commonly deleted major arc. The results confirmed that the content of cf-mtDNA in CSF was statistically significantly different between iPD and LRRK2-PD patients. Moreover, we found high cf-mtDNA deletion levels in CSF from patients with iPD, but not LRRK2-PD. The high cf-mtDNA deletion frequency in iPD was validated in an independent cohort. These results indicated that the content and deletion ratio of cf-mtDNA may differentiate iPD from LRRK2-PD, and provides further evidence of the different mitochondrial pathophysiology between these two forms of the disease.

Serum MicroRNAs Predict Isolated Rapid Eye Movement Sleep Behavior Disorder and Lewy Body Diseases.

BACKGROUND: Isolated rapid eye movement sleep behavior disorder (IRBD) is a well-established clinical risk factor for Lewy body diseases (LBDs), such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB). OBJECTIVE: To elucidate whether serum microRNA (miRNA) deregulation in IRBD can antedate the diagnosis of LBD by performing a longitudinal study in different progression stages of IRBD before and after LBD diagnosis and assessing the predictive performance of differentially expressed miRNAs by machine learning-based modeling. METHODS: Using genome-wide miRNA analysis and real-time quantitative polymerase chain reaction validation, we assessed serum miRNA profiles from patients with IRBD stratified by dopamine transporter (DaT) single-photon emission computed tomography into DaT-negative IRBD (n = 17) and DaT-positive IRBD (n = 21), IRBD phenoconverted into LBD (n = 13), and controls (n = 20). Longitudinally, we followed up the IRBD cohort by studying three time point serum samples over 26 months. RESULTS: We found sustained cross-sectional and longitudinal deregulation of 12 miRNAs across the RBD continuum, including DaT-negative IRBD, DaT-positive IRBD, and LBD phenoconverted IRBD (let-7c-5p, miR-19b-3p, miR-140, miR-22-3p, miR-221-3p, miR-24-3p, miR-25-3p, miR-29c-3p, miR-361-5p, miR-425-5p, miR-4505, and miR-451a) (false discovery rate P < 0.05). Age- and sex-adjusted predictive modeling based on the 12 differentially expressed miRNA biosignatures discriminated IRBD and PD or DLB from controls with an area under the curve of 98% (95% confidence interval: 89-99%). CONCLUSIONS: Besides clinical diagnosis of IRBD or imaging markers such as DaT single-photon emission computed tomography, specific miRNA biosignatures alone hold promise as progression biomarkers for patients with IRBD for predicting PD and DLB clinical outcomes. Further miRNA studies in other PD at-risk populations, such as LRRK2 mutation asymptomatic carriers or hyposmic subjects, are warranted. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Increased Phospho-AKT in Blood Cells from LRRK2 G2019S Mutation Carriers.

The purpose of this study was to investigate whether  differential phosphorylation states of blood markers can identify patients with LRRK2 Parkinson's disease (PD). We assessed phospho(P)-Ser-935-LRRK2 and P-Ser-473-AKT levels in peripheral blood cells from patients with G2019S LRRK2-associated PD (L2PD, n = 31), G2019S LRRK2 non-manifesting carriers (L2NMC, n = 26), idiopathic PD (iPD, n = 25), and controls (n = 40, total n = 122). We found no differences at P-Ser-935-LRRK2 between groups but detected a specific increase of P-Ser-473-AKT levels in all G2019S carriers, either L2PD or L2NMC, absent in iPD. Although insensitive to LRRK2 inhibition, our study identifies P-Ser-473-AKT as an endogenous candidate biomarker for peripheral inflammation in G2019S carriers using accessible blood cells. ANN NEUROL 2022;92:888-894.

Utility of Postoperative Imaging Software for Deep Brain Stimulation Targeting in Patients with Movement Disorders.

OBJECTIVE: The objective of this study was to evaluate the accuracy of the SureTune3 postoperative imaging software in determining the location of a deep brain stimulation (DBS) electrode based on clinical outcomes and the adverse effects (AEs) observed. METHODS: Twenty-six consecutive patients with Parkinson disease (n = 17), essential tremor (n = 8), and dystonia (n = 1) who underwent bilateral DBS surgery (52 electrodes) were included in this study. Presurgical assessments were performed in all patients prior to surgery and at 3 and 6 months after surgery, using quality-of-life and clinical scales in each case. The SureTune3 software was used to evaluate the anatomical positioning of the DBS electrodes. RESULTS: Following DBS surgery, motor and quality-of-life improvement was observed in all patients. Different AEs were detected in 12 patients, in 10 of whom (83.3%) SureTune3 related the symptoms to the positioning of an electrode. A clinical association was observed with SureTune3 for 48 of 52 (92.3%) electrodes, whereas no association was found between the AEs or clinical outcomes and the SureTune3 reconstructions for 4 of 52 electrodes (7.7%) from 4 different patients. In 2 patients, the contact chosen was modified based on the SureTune3 data, and in 2 cases, the software helped determine that second electrode replacement surgery was necessary. CONCLUSIONS: The anatomical position of electrodes analyzed with SureTune3 software was strongly correlated with both the AEs and clinical outcomes. Thus, SureTune3 may be useful in clinical practice, and it could help improve stimulation parameters and influence decisions to undertake electrode replacement surgery.

WHOPPA Enables Parallel Assessment of Leucine-Rich Repeat Kinase 2 and Glucocerebrosidase Enzymatic Activity in Parkinson's Disease Monocytes.

Both leucine-rich repeat kinase 2 (LRRK2) and glucocerebrosidase (GCase) are promising targets for the treatment of Parkinson's disease (PD). Evidence suggests that both proteins are involved in biological pathways involving the lysosome. However, studies to date have largely investigated the enzymes in isolation and any relationship between LRRK2 and GCase remains unclear. Both enzymes are highly expressed in peripheral blood monocytes and have been implicated in immune function and inflammation. To facilitate the standardized measurement of these readouts in large cohorts of samples collected from persons with PD across the globe, we developed and optimized a sample collection and processing protocol with parallel flow cytometry assays. Assay parameters were first optimized using healthy control peripheral blood mononuclear cells (PBMCs), and then LRRK2 and GCase activities were measured in immune cells from persons with idiopathic PD (iPD). We tested the ability of this protocol to deliver similar results across institutes across the globe, and named this protocol the Wallings-Hughes Optimized Protocol for PBMC Assessment (WHOPPA). In the application of this protocol, we found increased LRRK2 levels and stimulation-dependent enzymatic activity, and decreased GBA index in classical iPD monocytes, as well as increased cytokine release in PD PBMCs. WHOPPA also demonstrated a strong positive correlation between LRRK2 levels, pRab10 and HLA-DR in classical monocytes from subjects with iPD. These data support a role for the global use of WHOPPA and expression levels of these two PD-associated proteins in immune responses, and provide a robust assay to determine if LRRK2 and GCase activities in monocytes have potential utility as reliable and reproducible biomarkers of disease in larger cohorts of subjects with PD.

Over-Mutated Mitochondrial, Lysosomal and TFEB-Regulated Genes in Parkinson's Disease.

The association between Parkinson's disease (PD) and mutations in genes involved in lysosomal and mitochondrial function has been previously reported. However, little is known about the involvement of other genes or cellular mechanisms. We aim to identify novel genetic associations to better understand the pathogenesis of PD. We performed WES in a cohort of 32 PD patients and 30 age-matched controls. We searched for rare variants in 1667 genes: PD-associated, related to lysosomal function and mitochondrial function and TFEB-regulated. When comparing the PD patient cohort with that of age matched controls, a statistically significant burden of rare variants in the previous group of genes were identified. In addition, the Z-score calculation, using the European population database (GnomAD), showed an over-representation of particular variants in 36 genes. Interestingly, 11 of these genes are implicated in mitochondrial function and 18 are TFEB-regulated genes. Our results suggest, for the first time, an involvement of TFEB-regulated genes in the genetic susceptibility to PD. This is remarkable as TFEB factor has been reported to be sequestered inside Lewy bodies, pointing to a role of TFEB in the pathogenesis of PD. Our data also reinforce the involvement of lysosomal and mitochondrial mechanisms in PD.

Altered expression of the immunoregulatory ligand-receptor pair CD200-CD200R1 in the brain of Parkinson's disease patients.

Neuroinflammation, in which activated microglia are involved, appears to contribute to the development of Parkinson's disease (PD). However, the role of microglial activation and the mechanisms governing this process remain uncertain. We focused on one inhibitory mechanism involved in the control of microglial activation, the microglia inhibitory receptor CD200R1, and its ligand CD200, mainly expressed by neurons. The human CD200R1 gene encodes two membrane-associated and two soluble protein isoforms and the human CD200 gene encodes full-length proteins (CD200full) but also truncated (CD200tr) proteins which act as CD200R1 antagonists. Little is known about their expression in the human brain under pathological conditions. We used human peripheral blood monocytes and monocyte-derived microglia-like cells from control subjects to characterize the expression of the CD200R1 mRNA variants, which showed stimulus-specific responses. We provide evidence of increased CD200R1 (mRNA variants and protein isoforms) and CD200 expression (CD200tr mRNA) in brain tissue of PD patients, mainly in the hippocampus, as well as increased CD200 expression (CD200full and CD200tr mRNAs) in iPSCs-derived dopaminergic neurons generated from skin fibroblasts of PD patients. Our results suggest that CD200-CD200R1 signalling is altered in PD, which may affect the microglial function and constitute a potential target in therapeutic strategies for PD.

Differential Phospho-Signatures in Blood Cells Identify LRRK2 G2019S Carriers in Parkinson's Disease.

BACKGROUND: The clinicopathological phenotype of G2019S LRRK2-associated Parkinson's disease (L2PD) is similar to idiopathic Parkinson's disease (iPD), and G2019S LRRK2 nonmanifesting carriers (L2NMCs) are at increased risk for development of PD. With various therapeutic strategies in the clinical and preclinical pipeline, there is an urgent need to identify biomarkers that can aid early diagnosis and patient enrichment for ongoing and future LRRK2-targeted trials. OBJECTIVE: The objective of this work was to investigate differential protein and phospho-protein changes related to G2019S mutant LRRK2 in peripheral blood mononuclear cells from G2019S L2PD patients and G2019S L2NMCs, identify specific phospho-protein changes associated with the G2019S mutation and with disease status, and compare findings with patients with iPD. METHODS: We performed an unbiased phospho-proteomic study by isobaric label-based mass spectrometry using peripheral blood mononuclear cell group pools from a LRRK2 cohort from Spain encompassing patients with G2019S L2PD (n = 20), G2019S L2NMCs (n = 20), healthy control subjects (n = 30), patients with iPD (n = 15), patients with R1441G L2PD (n = 5), and R1441G L2NMCs (n = 3) (total N = 93). RESULTS: Comparing G2019S carriers with healthy controls, we identified phospho-protein changes associated with the G2019S mutation. Moreover, we uncovered a specific G2019S phospho-signature that changes with disease status and can discriminate patients with G2019S L2PD, G2019S L2NMCs, and healthy controls. Although patients with iPD showed a differential phospho-proteomic profile, biological enrichment analyses revealed similar changes in deregulated pathways across the three groups. CONCLUSIONS: We found a differential phospho-signature associated with LRRK2 G2019S for which, consistent with disease status, the phospho-profile from PD at-risk G2019S L2NMCs was more similar to healthy controls than patients with G2019S L2PD with the manifested disease. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Differentiation of multiple system atrophy subtypes by gray matter atrophy.

BACKGROUND AND PURPOSE: Multiple system atrophy(MSA) is a rare adult-onset synucleinopathy that can be divided in two subtypes depending on whether the prevalence of its symptoms is more parkinsonian or cerebellar (MSA-P and MSA-C, respectively). The aim of this work is to investigate the structural MRI changes able to discriminate MSA phenotypes. METHODS: The sample includes 31 MSA patients (15 MSA-C and 16 MSA-P) and 39 healthy controls. Participants underwent a comprehensive motor and neuropsychological battery. MRI data were acquired with a 3T scanner (MAGNETOM Trio, Siemens, Germany). FreeSurfer was used to obtain volumetric and cortical thickness measures. A Support Vector Machine (SVM) algorithm was used to assess the classification between patients' group using cortical and subcortical structural data. RESULTS: After correction for multiple comparisons, MSA-C patients had greater atrophy than MSA-P in the left cerebellum, whereas MSA-P showed reduced volume bilaterally in the pallidum and putamen. Using deep gray matter volume ratios and mean cortical thickness as features, the SVM algorithm provided a consistent classification between MSA-C and MSA-P patients (balanced accuracy 74.2%, specificity 75.0%, and sensitivity 73.3%). The cerebellum, putamen, thalamus, ventral diencephalon, pallidum, and caudate were the most contributing features to the classification decision (z > 3.28; p < .05 [false discovery rate]). CONCLUSIONS: MSA-C and MSA-P with similar disease severity and duration have a differential distribution of gray matter atrophy. Although cerebellar atrophy is a clear differentiator between groups, thalamic and basal ganglia structures are also relevant contributors to distinguishing MSA subtypes.

Non-motor symptoms in spasmodic dysphonia: A case control-study.

OBJECTIVE: Non-motor symptoms (NMS) have been identified in some focal adult-onset dystonia. In the present study we aimed to evaluate the presence of NMS in patients with spasmodic dysphonia (SD), a focal action-induced dystonia that affects intrinsic laryngeal muscle control. METHODS: Seventeen SD patients and 17 control subjects not significantly different in age and sex were evaluated for the presence of NMS. Additionally, voice handicap index (VHI-10), reflux symptom index, neuropsychiatric symptoms and QoL were assessed by validated scales and questionnaires. RESULTS: Patients' group significantly differed from control group in mild depressive symptoms (4.35 ± 3.9 vs. 1.47 ± 2; p=0.01), insomnia (35.3% vs. 14.7%; p=0.016), smell and taste loss (11.8% vs. 0%; p=0.033), swallowing difficulties (17.6% vs. 0%; p=0.007) and throat pain (17.6% vs. 0%; p=0.007). In the group of SD, there was no correlation between voice perception evaluated by VHI-10, number of NMS or QoL. CONCLUSION: Patients with SD have a greater burden of depressive, smell, taste, and sleep NMS than control subjects.

Disrupted functional connectivity in PD with probable RBD and its cognitive correlates.

Recent studies associated rapid eye movement sleep behavior disorder (RBD) in Parkinson's disease (PD) with severe cognitive impairment and brain atrophy. However, whole-brain functional connectivity has never been explored in this group of PD patients. In this study, whole-brain network-based statistics and graph-theoretical approaches were used to characterize resting-state interregional functional connectivity in PD with probable RBD (PD-pRBD) and its relationship with cognition. Our sample consisted of 30 healthy controls, 32 PD without probable RBD (PD-non pRBD), and 27 PD-pRBD. The PD-pRBD group showed reduced functional connectivity compared with controls mainly involving cingulate areas with temporal, frontal, insular, and thalamic regions (p < 0.001). Also, the PD-pRBD group showed reduced functional connectivity between right ventral posterior cingulate and left medial precuneus compared with PD-non pRBD (p < 0.05). We found increased normalized characteristic path length in PD-pRBD compared with PD-non pRBD. In the PD-pRBD group, mean connectivity strength from reduced connections correlated with visuoperceptual task and normalized characteristic path length correlated with processing speed and verbal memory tasks. This work demonstrates the existence of disrupted functional connectivity in PD-pRBD, together with abnormal network integrity, that supports its consideration as a severe PD subtype.

Prediabetes, type 2 diabetes mellitus and risk of Parkinson's disease: A population-based cohort study.

BACKGROUND: Association of type 2 diabetes mellitus (T2D) with subsequent Parkinson's disease (PD) has supported the link between glucose metabolism and PD. We assessed the risk of PD not only in T2D but also in prediabetes. METHODS: We conducted a retrospective cohort study of the population attended in primary care centres of the Catalan Health Institute in Catalonia between 2006 and 2018. The data were obtained from the Information System for Research in Primary Care (SIDIAP). We created a cohort of T2D and prediabetes patients (HbA1c ≥ 5.7-6.4% without antidiabetic drugs or previous T2D diagnosis) and compared to a reference cohort. The outcome was PD diagnosis and we excluded PD before or during the first year of follow-up. We used multivariate Cox regression models to calculate hazard ratios (HR) and 95% confidence intervals (95%CI). We excluded subjects with atypical and secondary parkinsonisms. RESULTS: The exposed cohorts comprised of 281.153 patients with T2D and 266.379 with prediabetes and a reference cohort of 2.556.928 subjects. T2D and prediabetes were associated with higher risk of PD (HRadjusted 1.19, 95%CI 1.13-1.25, and 1.07, 1.00-1.14; respectively). In analyses stratified by sex, prediabetes was only associated with PD risk in women (1.12, 1.03-1.22 vs. 1.01, 0.99-1.10 in men). When analysis was stratified by age, T2D and prediabetes were associated with a greater PD risk both in women (2.36, 1.96-2.84 and 2.10, 1.70-2.59 respectively) and men (1.74, 1.52-2.00 and 1.90, 1.57-2.30 respectively) below 65 years-old. CONCLUSIONS: We report for the first time that prediabetes increases the odds of subsequent PD and replicate the association with established T2D. Both associations predominate in women and young individuals.

R1441G but not G2019S mutation enhances LRRK2 mediated Rab10 phosphorylation in human peripheral blood neutrophils.

Heterozygous gain-of-kinase function variants in LRRK2 (leucine-rich repeat kinase 2) cause 1-2% of all cases of Parkinson's disease (PD) albeit with incomplete and age-dependent penetrance. All pathogenic LRRK2 mutations reside within the two catalytic domains of LRRK2-either in its kinase domain (e.g. G2019S) with modest effect or its ROC-COR GTPase domain (e.g. R1441G/H) with large effect on LRRK2 kinase activity. We have previously reported assays to interrogate LRRK2 kinase pathway activity in human bio-samples measuring phosphorylation of its endogenous substrate Rab10, that mirrors LRRK2 kinase activation status. Here, we isolated neutrophils from fresh peripheral blood from 101 participants including 42 LRRK2 mutation carriers (21 with the G2019S and 21 with the R1441G mutations), 27 patients with idiopathic PD, and 32 controls. Using a dual approach, LRRK2 dependent Rab10 phosphorylation at Threonine 73 (pRab10Thr73) was measured by quantitative multiplexed immunoblotting for pRab10Thr73/total Rab10 as well as targeted mass-spectrometry for absolute pRab10Thr73 occupancy. We found a significant over fourfold increase in pRab10Thr73 phosphorylation in carriers of the LRRK2 R1441G mutation irrespective of clinical disease status. The effect of the LRRK2 G2019S mutation did not reach statistical significance. Furthermore, we show that LRRK2 phosphorylation at Serine 935 is not a marker for LRRK2 kinase activity in human neutrophils. When analysing pRab10Thr73 phosphorylation in post-mortem brain samples, we observed overall high variability irrespective of clinical and LRRK2 mutation status and attributed this mainly to the adverse effect of the peri- and post-mortem period on the stability of posttranslational modifications such as protein phosphorylation. Overall, in vivo LRRK2 dependent pRab10Thr73 phosphorylation in human peripheral blood neutrophils is a specific, robust and promising biomarker for significant LRRK2 kinase hyperactivation, as with the LRRK2 R1441G mutation. Additional readouts and/or assays may be needed to increase sensitivity to detect modest LRRK2 kinase activation, as with the LRRK2 G2019S mutation. Our assays could be useful for patient stratification and target engagement studies for LRRK2 kinase inhibitors.

Regulatory rare variants of the dopaminergic gene ANKK1 as potential risk factors for Parkinson's disease.

Parkinson's disease (PD) is characterized by cerebral dopamine depletion that causes motor and cognitive deficits. The dopamine-related gene ANKK1 has been associated with neuropsychiatric disorders with a dopaminergic deficiency in the striatum. This study aims to define the contribution of ANKK1 rare variants in PD. We found in 10 out of 535 PD patients 6 ANKK1 heterozygous rare alleles located at the 5'UTR, the first exon, intron 1, and the nearby enhancer located 2.6 kb upstream. All 6 ANKK1 single nucleotide variants were located in conserved regulatory regions and showed significant allele-dependent effects on gene regulation in vitro. ANKK1 variant carriers did not show other PD-causing Mendelian mutations. Nevertheless, four patients were heterozygous carriers of rare variants of ATP7B gene, which is related to catecholamines. We also found an association between the polymorphic rs7107223 of the ANKK1 enhancer and PD in two independent clinical series (P = 0.007 and 0.021). rs7107223 functional analysis showed significant allele-dependent effects on both gene regulation and dopaminergic response. In conclusion, we have identified in PD patients functional variants at the ANKK1 locus highlighting the possible relevance of rare variants and non-coding regulatory regions in both the genetics of PD and the dopaminergic vulnerability of this disease.