Insights into the Value of Lyso-Gb1 as a Predictive Biomarker in Treatment-Naïve Patients with Gaucher Disease Type 1 in the LYSO-PROOF Study.

Gaucher disease (GD) is a rare autosomal recessive disorder arising from bi-allelic variants in the GBA1 gene, encoding glucocerebrosidase. Deficiency of this enzyme leads to progressive accumulation of the sphingolipid glucosylsphingosine (lyso-Gb1). The international, multicenter, observational "Lyso-Gb1 as a Long-term Prognostic Biomarker in Gaucher Disease"-LYSO-PROOF study succeeded in enrolling a cohort of 160 treatment-naïve GD patients from diverse geographic regions and evaluated the potential of lyso-Gb1 as a specific biomarker for GD. Using genotypes based on established classifications for clinical presentation, patients were stratified into type 1 GD (n = 114) and further subdivided into mild (n = 66) and severe type 1 GD (n = 48). Due to having previously unreported genotypes, 46 patients could not be classified. Though lyso-Gb1 values at enrollment were widely distributed, they displayed a moderate and statistically highly significant correlation with disease severity measured by the GD-DS3 scoring system in all GD patients (r = 0.602, p < 0.0001). These findings support the utility of lyso-Gb1 as a sensitive biomarker for GD and indicate that it could help to predict the clinical course of patients with undescribed genotypes to improve personalized care in the future.

Quantitation of a Urinary Profile of Biomarkers in Gaucher Disease Type 1 Patients Using Tandem Mass Spectrometry.

Gaucher disease is a rare inherited disorder caused by a deficiency of the lysosomal acid beta-glucocerebrosidase enzyme. Metabolomic studies by our group targeted several new potential urinary biomarkers. Apart from lyso-Gb1, these studies highlighted lyso-Gb1 analogs -28, -26, -12 (A/B), +2, +14, +16 (A/B), +30, and +32 Da, and polycyclic lyso-Gb1 analogs 362, 366, 390, and 394 Da. The main objective of the current study was to develop and validate a robust UPLC-MS/MS method to study the urine distribution of these biomarkers in patients. METHOD: Urine samples were purified using solid-phase extraction. A 12 min UPLC-MS/MS method was developed. RESULTS: Validation assays revealed high precision and accuracy for creatinine and lyso-Gb1. Most lyso-Gb1 analogs had good recovery rates and high intra- and interday precision assays. Biomarker-estimated LOD and LOQ levels ranged from 56-109 pM to 186-354 pM, respectively. Comparison between GD patients and healthy controls showed significant differences in most biomarker levels. Typically, treated GD patients presented lower biomarker levels compared to untreated patients. CONCLUSIONS: These data suggest that the metabolites investigated might be interesting GD biomarkers. More studies with a larger cohort of patients will be needed to better understand the clinical significance of these GD biomarkers.

Metabolomic Study Using Time-of-Flight Mass Spectrometry Reveals Novel Urinary Biomarkers for Gaucher Disease Type 1.

Gaucher disease (GD) is a lysosomal storage disorder resulting from a biallelic mutation in the gene GBA1, leading to deficiencies in the enzyme ß-glucocerebrosidase (Gcase). Inabilities of the Gcase to catabolize its substrate result in the accumulation of sphingolipids in macrophages, which impairs the cell functions and ultimately leads to multisystemic clinical manifestations. Important variability in symptoms and manifestations may lead to challenging diagnosis and patient care. Plasma glucosylsphingosine (lyso-Gb1) is a biomarker frequently used for prognosis, monitoring, and patient follow-up. While lyso-Gb1 appears to be a valid biomarker, few studies have investigated other matrices for potential GD biomarkers. The main objective of this study was to investigate the urine matrix as a potential source of new GD biomarkers by performing a metabolomic study using time-of-flight mass spectrometry. Our study highlighted a significant increase of eight urinary lyso-Gb1 analogues. Moreover, a novel class of biomarkers, named polycyclic lyso-Gb1 analogues, was identified. These four new molecules were more elevated than lyso-Gb1 and related analogues in urine specimens of GD patients. Further investigations are warranted to validate the efficiency of these newly found biomarkers on a larger cohort of Gaucher patients and to compare them with plasma biomarkers currently quantified in clinical laboratories.

Quantitation of a plasma biomarker profile for the early detection of Gaucher disease type 1 patients.

Aim: Gaucher disease (GD) is caused by a deficiency of the lysosomal enzyme acid ß-glucocerebrosidase. Recent metabolomic studies highlighted several new metabolites increased in the plasma of GD patients. We aimed to develop and validate a UPLC-MS/MS method allowing a relative quantitation of lyso-Gb1 and lyso-Gb1 analogs -28, -12, -2, +14, +16 and +18 Da in addition to sphingosylphosphorylcholine, N-palmitoyl-O-phosphocholine to study potential correlations with clinical manifestations. Methodology & results: Following solid-phase extraction, plasma samples were evaporated and resuspended in 100 µl of resuspension solution. Three microliter is injected into the UPLC-MS/MS for analysis. Conclusion: All biomarkers studied were increased in GD patients. Significant correlations were observed between specific analogs and hematological, and visceral complications, as well as overall disease severity.

Lysosphingolipid urine screening test using mass spectrometry for the early detection of lysosomal storage disorders.

Background: Sphingolipidoses are caused by a defective sphingolipid catabolism, leading to an accumulation of several glycolipid species in tissues and resulting in neurotoxicity and severe systemic manifestations. Methods & results: Urine samples from controls and patients were purified by solid-phase extraction prior to the analysis by ultra-high-performance liquid chromatography (UPLC) combined with MS/MS. A UPLC-MS/MS method for the analysis of 21 urinary creatinine-normalized biomarkers for eight diseases was developed and validated. Conclusion: Considering the growing demand to identify patients with different sphingolipidoses early and reliably, this methodology will be applied for high-risk screening to target efficiently patients with various sphingolipidoses.

The Rostock International Parkinson's Disease (ROPAD) Study: Protocol and Initial Findings.

BACKGROUND: Genetic stratification of Parkinson's disease (PD) patients facilitates gene-tailored research studies and clinical trials. The objective of this study was to describe the design of and the initial data from the Rostock International Parkinson's Disease (ROPAD) study, an epidemiological observational study aiming to genetically characterize ~10,000 participants. METHODS: Recruitment criteria included (1) clinical diagnosis of PD, (2) relative of participant with a reportable LRRK2 variant, or (3) North African Berber or Ashkenazi Jew. DNA analysis involved up to 3 successive steps: (1) variant (LRRK2) and gene (GBA) screening, (2) panel sequencing of 68 PD-linked genes, and (3) genome sequencing. RESULTS: Initial data based on the first 1360 participants indicated that the ROPAD enrollment strategy revealed a genetic diagnostic yield of ~14% among a PD cohort from tertiary referral centers. CONCLUSIONS: The ROPAD screening protocol is feasible for high-throughput genetic characterization of PD participants and subsequent prioritization for gene-focused research efforts and clinical trials. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Rapid Large-Scale COVID-19 Testing During Shortages.

The Coronavirus disease 2019 (COVID-19) pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has resulted in economic and social lockdowns in most countries all over the globe. Early identification of infected individuals is regarded as one of the most important prerequisites for fighting the pandemic and for returning to a 'New Normal'. Large-scale testing is therefore crucial, but is facing several challenges including shortage of sample collection tools and of molecular biological reagents, and the need for safe electronic communication of medical reports. We present the successful establishment of a holistic SARS-CoV-2 testing platform that covers proband registration, sample collection and shipment, sample testing, and report issuing. The RT-PCR-based virus detection, being central to the platform, was extensively validated: sensitivity and specificity were defined as 96.8% and 100%, respectively; intra-run and inter-run precision were <3%. A novel type of sample swab and an in-house-developed RNA extraction system were shown to perform as good as commercially available products. The resulting flexibility guarantees independence from the current bottlenecks in SARS-CoV-2 testing. Based on our technology, we offered testing at local, national, and global levels. In the present study, we report the results from approx. 18,000 SARS-CoV-2 tests in almost 10,000 individuals from a low-frequency SARS-CoV-2 pandemic area in a homogenous geographical region in north-eastern Germany for a period of 10 weeks (21 March to 31 May 2020). Among the probands, five SARS-CoV-2 positive cases were identified. Comparative analysis of corresponding virus genomes revealed a diverse origin from three of the five currently recognized SARS-CoV-2 phylogenetic clades. Our study exemplifies how preventive SARS-CoV-2 testing can be set up in a rapid and flexible manner. The application of our test has enabled a safe maintenance/resume of critical local infrastructure, e.g., nursing homes where more than 5000 elderlies and caretakers got tested. The strategy outlined by the present study may serve as a blueprint for the implementation of large-scale preventive SARS-CoV-2 testing elsewhere.

Regulation of podosome formation in aortic endothelial cells vessels by physiological extracellular cues.

Invadosomes are specialised actin-based dynamic microdomains of the plasma membrane. Their occurrence has been associated with cell adhesion, matrix degrading and mechanosensory functions that make them crucial regulators of cell migration and invasion. Monocytic, cancer cell and Src-transformed cell invadosomes have been extensively described. Less well defined are the structures which form in other cell types, i.e., non-haematopoietic and non-transformed cells, exposed to specific stimuli. We herein describe the specificities of podosomes induced in aortic endothelial cells stimulated with TGFß in vitro and in conditions that more closely resemble the in vivo situation. These podosomes display the typical architecture of monocytic podosomes. They organise into large rosette-shape superstructures where they exhibit collective dynamic behavior consisting in cycles of formation and regression. At the ultrastructural level, microfilament arrangements in individual podosomes were revealed. Oxygen levels and hemodynamic forces, which are key players in endothelial cell biology, both influence the process. In 3D environment, podosomes appear as globular structures along cellular extensions. A better characterization of endothelial podosomes has far-reaching implications in the understanding and, possibly, in the treatment of some vascular diseases.

A Rho signaling network links microtubules to PKD controlled carrier transport to focal adhesions.

Protein kinase D (PKD) is a family of serine/threonine kinases that is required for the structural integrity and function of the Golgi complex. Despite its importance in the regulation of Golgi function, the molecular mechanisms regulating PKD activity are still incompletely understood. Using the genetically encoded PKD activity reporter G-PKDrep we now uncover a Rho signaling network comprising GEF-H1, the RhoGAP DLC3, and the Rho effector PLCε that regulate the activation of PKD at trans-Golgi membranes. We further show that this molecular network coordinates the formation of TGN-derived Rab6-positive transport carriers delivering cargo for localized exocytosis at focal adhesions.

ALK5 and ALK1 play antagonistic roles in transforming growth factor ß-induced podosome formation in aortic endothelial cells.

Transforming growth factor ß (TGF-ß) and related cytokines play a central role in the vascular system. In vitro, TGF-ß induces aortic endothelial cells to assemble subcellular actin-rich structures specialized for matrix degradation called podosomes. To explore further this TGF-ß-specific response and determine in which context podosomes form, ALK5 and ALK1 TGF-ß receptor signaling pathways were investigated in bovine aortic endothelial cells. We report that TGF-ß drives podosome formation through ALK5 and the downstream effectors Smad2 and Smad3. Concurrent TGF-ß-induced ALK1 signaling mitigates ALK5 responses through Smad1. ALK1 signaling induced by BMP9 also antagonizes TGF-ß-induced podosome formation, but this occurs through both Smad1 and Smad5. Whereas ALK1 neutralization brings ALK5 signals to full potency for TGF-ß-induced podosome formation, ALK1 depletion leads to cell disturbances not compatible with podosome assembly. Thus, ALK1 possesses passive and active modalities. Altogether, our results reveal specific features of ALK1 and ALK5 signaling with potential clinical implications.