Search

Mutations in GBA1, encoding glucocerebrosidase beta 1 (GCase), are the most common genetic risk factor for Parkinson's disease (PD). GCase dysfunction leads to an accumulation of glucosylceramide (GluCer) substrates in different organs and fluids. Despite the challenges in quantifying GluCer isoforms in biological samples, their potential clinical interest as PD biomarkers justifies the development of robust assays. An extensively evaluated high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for quantifying 14 GluCer and galactosylceramide (GalCer) isoforms in human cerebrospinal fluid (CSF) samples is presented. Sample pretreatment, HPLC, and MS/MS parameters were optimized. Evaluation was performed according to the recommendations of the Clinical and Laboratory Standards Institute and European Medicines Agency guidelines. Four 7-point calibration curves were generated, with a linearity interval from 2.5 to 200 nM ( R 2 ≥ 0.995). The limit of quantification was set at 5 nM. Between-run precision and accuracy were up to 12.5 and 9%, respectively. After method validation, we measured the levels of GluCer and GalCer isoforms in CSF human samples, including 6 healthy controls (HC), 22 idiopathic GBA1 wild-type PD (iPD) patients, and 5 GBA1-associated PD (PD-GBA) patients. GluCer/GalCer median ratios were found to be higher in the CSF of PD-GBA patients, particularly in severe GBA1 mutations, than those in iPD and HC. The observed trends in GluCer/GalCer ratios among groups provide novel information for the comprehensive analysis of sphingolipids as potential biomarkers of PD.

Objective: To assess whether aspirin treatment can be discontinued in pregnancies with normal uterine artery pulsatility index (≤90th percentile) at 24-28 weeks., Design: Post-hoc analysis of a clinical trial., Setting: Nine maternity hospitals in Spain., Population or Sample: Pregnant individuals at high risk of pre-eclampsia at 11-13 weeks and normal uterine artery Doppler at 24-28 weeks., Methods: All participants received treatment with daily aspirin at a dose of 150 mg. Participants were randomly assigned, in a 1:1 ratio, either to continue aspirin treatment until 36 weeks (control group) or to discontinue aspirin treatment (intervention group), between September 2019 and September 2021. In this secondary analysis, women with a UtAPI >90th percentile at 24-28 weeks were excluded. The non-inferiority margin was set at a difference of 1.9% for the incidence of preterm pre-eclampsia., Main Outcome Measures: Incidence of preterm pre-eclampsia., Results: Of the 1611 eligible women, 139 were excluded for UtAPI >90th percentile or if UtAPI was not available. Finally, 804 were included in this post-hoc analysis. Preterm pre-eclampsia occurred in three of 409 (0.7%) women in the aspirin discontinuation group and five of 395 (1.3%) women in the continuation group (-0.53; 95% CI -1.91 to 0.85), indicating non-inferiority of aspirin discontinuation., Conclusions: Discontinuing aspirin treatment at 24-28 weeks in women with a UtAPI ≤90th percentile was non-inferior to continuing aspirin treatment until 36 weeks for preventing preterm pre-eclampsia., (© 2023 The Authors. BJOG: An International Journal of Obstetrics and Gynaecology published by John Wiley & Sons Ltd.)

Lysosomal storage disorders (LSD) are a group of inherited metabolic diseases mainly caused by a deficiency of lysosomal hydrolases, resulting in a gradual accumulation of non-degraded substrates in different tissues causing the characteristic clinical manifestations of such disorders. Confirmatory tests of suspected LSD individuals include enzymatic and genetic testing. A well-oriented clinical suspicion can improve the cost-effectiveness of confirmatory tests and reduce the time expended to achieve the diagnosis. Thus, this work aims to retrospectively study the influence of clinical orientation on the diagnostic yield of enzymatic tests in LSD by retrieving clinical, biochemical, and genetic data obtained from subjects with suspicion of LSD. Our results suggest that the clinical manifestations at the time of diagnosis and the initial clinical suspicion can have a great impact on the diagnostic yield of enzymatic tests, and that clinical orientation performed in specialized clinical departments can contribute to improve it. In addition, the analysis of enzymatic tests as the first step in the diagnostic algorithm can correctly guide subsequent confirmatory genetic tests, in turn increasing their diagnostic yield. In summary, our results suggest that initial clinical suspicion plays a crucial role on the diagnostic yield of confirmatory enzymatic tests in LSD., Competing Interests: Conflict of interest All authors declare no conflict of interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Mutations in the GBA gene that encodes the lysosomal enzyme β-glucocerebrosidase (GCase) are a major genetic risk factor for Parkinson's disease (PD). In this study, we generated a set of differentiated and stable human dopaminergic cell lines that express the two most prevalent GBA mutations as well as GBA knockout cell lines as a in vitro disease modeling system to study the relationship between mutant GBA and the abnormal accumulation of α-synuclein. We performed a deep analysis of the consequences triggered by the presence of mutant GBA protein and the loss of GCase activity in different cellular compartments, focusing primarily on the lysosomal compartment, and analyzed in detail the lysosomal activity, composition, and integrity. The loss of GCase activity generates extensive lysosomal dysfunction, promoting the loss of activity of other lysosomal enzymes, affecting lysosomal membrane stability, promoting intralysosomal pH changes, and favoring the intralysosomal accumulation of sphingolipids and cholesterol. These local events, occurring only at a subcellular level, lead to an impairment of autophagy pathways, particularly chaperone-mediated autophagy, the main α-synuclein degradative pathway. The findings of this study highlighted the role of lysosomal function and lipid metabolism in PD and allowed us to describe a molecular mechanism to understand how mutations in GBA can contribute to an abnormal accumulation of different α-synuclein neurotoxic species in PD pathology., (© 2022. The Author(s).)

Predicting disease severity in patients infected with SARS-CoV-2 is difficult. Soluble angiotensin-converting enzyme 2 (sACE2) arises from the shedding of membrane ACE2 (mACE2), which is a receptor for SARS-CoV-2 spike protein. We evaluated the predictive value of sACE2 compared with known biomarkers of inflammation and tissue damage (CRP, GDF-15, IL-6, and sFlt-1) in 850 patients with and without SARS-CoV-2 with different clinical outcomes. For univariate analyses, median differences between biomarker levels were calculated for the following patient groups (classified by clinical outcome): RT-PCR-confirmed SARS-CoV-2 positive (Groups 1−4); RT-PCR-confirmed SARS-CoV-2 negative following previous SARS-CoV-2 infection (Groups 5 and 6); and ‘SARS-CoV-2 unexposed’ patients (Group 7). Median levels of CRP, GDF-15, IL-6, and sFlt-1 were significantly higher in hospitalized patients with SARS-CoV-2 compared with discharged patients (all p < 0.001), whereas levels of sACE2 were significantly lower (p < 0.001). ROC curve analysis of sACE2 provided cut-offs for predicting hospital admission (≤0.05 ng/mL (positive predictive value: 89.1%) and ≥0.42 ng/mL (negative predictive value: 84.0%)). These findings support further investigation of sACE2, as a single biomarker or as part of a panel, to predict hospitalization risk and disease severity in patients with SARS-CoV-2 infection.

Background: Different forms of pregenomic and other hepatitis B virus (HBV) RNA have been detected in patients' sera. These circulating HBV-RNAs may be useful for monitoring covalently closed circular DNA activity, and predicting hepatitis B e-antigen seroconversion or viral rebound after nucleos(t)ide analog cessation. Data on serum HBV-RNA quasispecies, however, is scarce. It is therefore important to develop methodologies to thoroughly analyze this quasispecies, ensuring the elimination of any residual HBV-DNA. Studying circulating HBV-RNA quasispecies may facilitate achieving functional cure of HBV infection., Aim: To establish a next-generation sequencing (NGS) methodology for analyzing serum HBV-RNA and comparing it with DNA quasispecies., Methods: Thirteen untreated chronic hepatitis B patients, showing different HBV-genotypes and degrees of severity of liver disease were enrolled in the study and a serum sample with HBV-DNA > 5 Log 10 IU/mL and HBV-RNA > 4 Log 10 copies/mL was taken from each patient. HBV-RNA was treated with DNAse I to remove any residual DNA, and the region between nucleotides (nt) 1255-1611 was amplified using a 3-nested polymerase chain reaction protocol, and analyzed with NGS. Variability/conservation and complexity was compared between HBV-DNA and RNA quasispecies., Results: No HBV-DNA contamination was detected in cDNA samples from HBV-RNA quasispecies. HBV quasispecies complexity showed heterogeneous behavior among patients. The Rare Haplotype Load at 1% was greater in DNA than in RNA quasispecies, with no statistically significant differences ( P = 0.1641). Regarding conservation, information content was equal in RNA and DNA quasispecies in most nt positions [218/357 (61.06%)]. In 102 of the remaining 139 (73.38%), HBV-RNA showed slightly higher variability. Sliding window analysis identified 4 hyper-conserved sequence fragments in each quasispecies, 3 of them coincided between the 2 quasispecies: nts 1258-1286, 1545-1573 and 1575-1604. The 2 hyper-variable sequence fragments also coincided: nts 1311-1344 and 1461-1485. Sequences between nts 1519-1543 and 1559-1587 were only hyper-conserved in HBV-DNA and RNA, respectively., Conclusion: Our methodology allowed analyzing HBV-RNA quasispecies complexity and conservation without interference from HBV-DNA. Thanks to this, we have been able to compare both quasispecies in the present study., Competing Interests: Conflict-of-interest statement: Authors have no conflict of interest for this manuscript., (©The Author(s) 2021. Published by Baishideng Publishing Group Inc. All rights reserved.)