False negatives in GBA1 sequencing due to polymerase dependent allelic imbalance

A variant in the GBA1 gene is one of the most common genetic risk factors to develop Parkinson's disease (PD). Here the serendipitous finding is reported of a polymerase dependent allelic imbalance when using next generation sequencing, potentially resulting in false-negative results when the allele frequency falls below the variant calling threshold (by default commonly at 30%). The full GBA1 gene was sequenced using next generation sequencing on saliva derived DNA from PD patients. Four polymerase chain reaction conditions were varied in twelve samples, to investigate the effect on allelic imbalance: (1) the primers (n=4); (2) the polymerase enzymes (n=2); (3) the primer annealing temperature (T-a) specified for the used polymerase; and (4) the amount of DNA input. Initially, 1295 samples were sequenced using Q5 High-Fidelity DNA Polymerase. 112 samples (8.6%) had an exonic variant and an additional 104 samples (8.0%) had an exonic variant that did not pass the variant frequency calling threshold of 30%. After changing the polymerase to TaKaRa LA Taq DNA Polymerase Hot-Start Version: RR042B, all samples had an allele frequency passing the calling threshold. Allele frequency was unaffected by a change in primer, annealing temperature or amount of DNA input. Sequencing of the GBA1 gene using next generation sequencing might be susceptible to a polymerase specific allelic imbalance, which can result in a large amount of flase-negative results. This was resolved in our case by changing the polymerase. Regions displaying low variant calling frequencies in GBA1 sequencing output in previous and future studies might warrant additional scrutiny.Perioperative Medicine: Efficacy, Safety and Outcome (Anesthesiology/Intensive Care

A biomarker study in patients with GBA1-parkinson's disease and healthy controls

BackgroundMolecules related to glucocerebrosidase (GCase) are potential biomarkers for development of compounds targeting GBA1-associated Parkinson's disease (GBA-PD).ObjectivesAssessing variability of various glycosphingolipids (GSLs) in plasma, peripheral blood mononuclear cells (PBMCs), and cerebrospinal fluid (CSF) across GBA-PD, idiopathic PD (iPD), and healthy volunteers (HVs).MethodsData from five studies were combined. Variability was assessed of glucosylceramide (various isoforms), lactosylceramide (various isoforms), glucosylsphingosine, galactosylsphingosine, GCase activity (using fluorescent 4-methylumbeliferryl-β-glucoside), and GCase protein (using enzyme-linked immunosorbent assay) in plasma, PBMCs, and CSF if available, in GBA-PD, iPD, and HVs. GSLs in leukocyte subtypes were compared in HVs. Principal component analysis was used to explore global patterns in GSLs, clinical characteristics (Movement Disorder Society – Unified Parkinson's Disease Rating Scale Part 3 [MDS-UPDRS-3], Mini-Mental State Examination [MMSE], GBA1 mutation type), and participant status (GBA-PD, iPD, HVs).ResultsWithin-subject between-day variability ranged from 5.8% to 44.5% and was generally lower in plasma than in PBMCs. Extracellular glucosylceramide levels (plasma) were slightly higher in GBA-PD compared with both iPD and HVs, while intracellular levels were comparable. GSLs in the different matrices (plasma, PBMCs, CSF) did not correlate. Both lactosylceramide and glucosylsphingosine were more abundant in granulocytes compared with monocytes and lymphocytes. Absolute levels of GSL isoforms differed greatly. GBA1 mutation types could not be differentiated based on GSL data.ConclusionsGlucosylceramide can stably be measured over days in both plasma and PBMCs and may be used as a biomarker in clinical trials targeting GBA-PD. Glucosylsphingosine and lactosylceramide are stable in plasma but are strongly affected by leukocyte subtypes in PBMCs. GBA-PD could be differentiated from iPD and HVs, primarily based on glucosylceramide levels in plasma. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.Perioperative Medicine: Efficacy, Safety and Outcome (Anesthesiology/Intensive Care

A randomized single and multiple ascending dose study in healthy volunteers of LTI-291, a centrally penetrant glucocerebrosidase activator

Aims A mutation in the GBA1 gene is the most common genetic risk factor for developing Parkinson's disease. GBA1 encodes the lysosomal enzyme glucosylceramidase beta (glucocerebrosidase, GCase) and mutations decrease enzyme activity. LTI-291 is an allosteric modulator of GCase, enhancing its activity. These first-in-human studies evaluated the safety, tolerability, pharmacokinetics and pharmacodynamics of single and multiple ascending doses of LTI-291 in healthy volunteers.Methods In the single ascending dose (SAD) study, 40 healthy volunteers were randomly assigned to LTI-291 (n = 8 per dose level) or placebo (n = 2 per dose level). Single doses of 3, 10, 30 and 90 mg LTI-291 were investigated. In the multiple ascending dose (MAD) study, 40 healthy middle-aged or elderly volunteers were randomly assigned to LTI-291 (n = 8 per dose level) or placebo (n = 2 per dose level). Fourteen consecutive daily doses of 3, 10, 30 and 60 mg LTI-291 or placebo were administered. In both the SAD and MAD studies, glycosphingolipid levels were measured and a test battery of neurocognitive tasks was performed.Results LTI-291 was generally well tolerated and no deaths or treatment-related SAEs occurred and no subject withdrew from a study due to AEs. C-max, AUC(0-24) and AUC(0-inf) increased in a dose proportional manner. The median half-life was 28.0 hours after multiple dosing. No dose-dependent glycosphingolipid changes occurred. No neurocognitive adverse effects were detected.Conclusions These first-in-human studies demonstrated that LTI-291 was well tolerated when given orally once daily for 14 consecutive days. This supports the continued clinical development and the exploration of LTI-291 effects in a GBA1-mutated Parkinson population.Perioperative Medicine: Efficacy, Safety and Outcome (Anesthesiology/Intensive Care

A Large-Scale Full GBA1 Gene Screening in Parkinson's Disease in the Netherlands

Background: The most common genetic risk factor for Parkinson’s disease known is a damaging variant in the GBA1 gene. The entire GBA1 gene has rarely been studied in a large cohort from a single population. The objective of this study was to assess the entire GBA1 gene in Parkinson’s disease from a single large population. Methods: The GBA1 gene was assessed in 3402 Dutch Parkinson’s disease patients using nextgeneration sequencing. Frequencies were compared with Dutch controls (n = 655). Family history of Parkinson’s disease was compared in carriers and noncarriers. Results: Fifteen percent of patients had a GBA1 nonsynonymous variant (including missense, frameshift, and recombinant alleles), compared with 6.4% of c

Substrate specificity of tissue-type and urokinase-type plasminogen activators

Recent studies suggest that plasminogen activators not only hydrolyse a specific arginine-valine bond in plasminogen, but may also cleave other proteins such as fibronectin. We studied the substrate specificity, particularly the preference for arginyl over lysyl peptide bonds, of tissue-type plasminogen activator (t-PA) as well as of two-chain urokinase-type plasminogen activator (u-PA). The arginine/lysine preference was determined with three pairs of tripeptidyl-p-nitroanilide substrates having either arginine or lysine in the P1 position and varied from 5.2 to 14.1 for u-PA and from 55.6 to 99.8 for t-PA. It was concluded that both t-PA and u-PA preferred arginyl to lysyl peptide bonds. However, u-PA had a significantly lower arginine/lysine preference than t-PA, indicating that u-PA represents a less specific proteinase. This may point to functions of u-PA other than plasminogen activation, which involve cleavage of lysyl bonds

Characterization of the interaction between urinary urokinase and the asialoglycoprotein receptor of liver cells

Urokinase-type plasminogen activator (u-PA) is rapidly cleared from the circulation with a half-life of a few minutes. We have previously shown that non-glycosylated recombinant u-PA is recognized by LRP, whereas urinary u-PA is recognized by the asialoglycoprotein receptor (ASGPr) on liver parenchymal cells. In the present study we biochemically characterized the latter interaction. The ASGPr was isolated and purified from rat liver tissue and a binding assay was developed. In this assay urinary uPA specifically bound to the receptor with apparent Kd ≈ 30 nM. Specific antibodies against the receptor completely blocked the binding of u-PA. In line with the known carbohydrate specificity of the ASGPr, D-GalNAc proved to be the most effective inhibitor of u-PA binding from a series of monosaccharides, followed by D-Gal and L-Fuc, whereas D-GlcNAc was ineffective. Recent literature showed that the N-linked oligosaccharides of urinary u-PA do not terminate with the common Gal-GlcNAc element but with a GalNAc-GlcNAc element, which is partially sulfated (Eur. J. Biochem. 1995, 228:1009). Sulfated forms of u-PA were separated from non-sulfated forms by using the lectin Wisteria floribunda agglutinin. Only the non-sulfated forms of u-PA (30 % of the total) appeared to bind to the ASGPr. We conclude that a fraction of urinary u-PA bears oligosaccharides which are specifically recognized by the asialoglycoprotein receptor on liver cells