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Inspecting VWF gene deep intronic region and in-depth study of the endothelial colony-forming cells to identify an underlying pathogenic molecular mechanism in a type 3 VWD patient

ABSTRACT: A type 3 von Willebrand disease (VWD) index patient (IP) remains mutation-negative after completion of conventional diagnostic analysis, including multiplex ligation-dependent probe amplification and sequencing of the promotor, exons, and flanking intronic regions of VWF gene (VWF). In this study, we intended to elucidate causitive genetic defect through screening of the whole VWF (including complete intronic region), mRNA analysis, and study of the patient-derived endothelial colony-forming cells (ECFCs). The entire VWF was analyzed by next-generation sequencing (NGS) on an Illumina platform. The NGS revealed a novel variant in VWF intron 8 (997+118 T>G). The subsequent assessments using bioinformatics tools (e.g. SpliceAl) predicted this variant creates a new donor splice site (ss) in intron 8, which could outcompete the consensus 5’ donor ss at exon/intron 8 junction. This leads to an aberrant mRNA which contains a premature stop codon, targeting it to nonsense-mediated mRNA decay. The VWF mRNA from whole blood and isolated ECFCs were quantified using the TaqMan assay on an ABI 7500 real-time PCR system. The quantitative analysis confirmed the virtual absence of VWF mRNA. Additionally, the level of secreted VWF from IP ECFCs was considerably reduced (~6% of healthy donors).

ORGANISM(S): Homo sapiens

PROVIDER: GSE195695 | GEO | 2022/03/30

REPOSITORIES: GEO

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Multifaceted Pathomolecular Mechanism of a VWF Large Deletion Involved in the Pathogenesis of Severe VWD

Project description:An in-frame heterozygous large deletion of exons 4-34 of the von Willebrand factor (VWF) gene was identified in an index patient (IP) with type 3 von Willebrand disease (VWD), as the only mutation. The IP exhibited severe bleeding episodes despite prophylaxis treatment, with a short VWF half-life after infusion of VWF/FVIII concentrates. This study intends to elucidate the causal molecular mechanism of this large deletion. Transcript analysis confirmed transcription of normal VWF mRNA besides an aberrant deleted transcript. The amount of secreted VWF from blood outgrowth endothelial cells (BOECs) isolated from the IP was not significantly different from that of controls. However, IP-BOECs exhibited a deficiency in the assembly of VWF multimers and biogenesis of the Weibel-Palade bodies (WPBs). Furthermore, immunostaining of IP-BOECs demonstrated subcellular mislocalization of WPBs pro-inflammatory cargos angiopoietin-2 (Ang2) and P-selectin. Additionally, whole-transcriptome RNA-sequencing of the BOECs and subsequent Ingenuity Pathway Analysis indicated the significant alterations of canonical pathways in IP-BOECs related to inflammatory responses, cell adhesion, extracellular organization, and angiogenesis (e.g. granulocyte adhesion and Rho-related signaling pathways), which are known downstream signaling pathways induced by Ang2. Accordingly, the IP-BOECs exhibited an increased adhesiveness to leukocytes which may contribute to accelerated VWF clearance. In conclusion, deleted VWF has a dominant-negative impact on the elongation of multimers and biogenesis of WPBs. Aberrant WPBs lead to the alternative trafficking of its cargos in a specific way, which, in turn, may cause distinctive perturbations in cellular signaling pathways, resulting in the exceptional phenotypes in the current patient.

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Prospective surface marker-based isolation and expansion of fetal endothelial colony-forming cells from human term placenta.

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Project description:Prp2 (U2AF65) is an essential splicing factor, that recognizes the poly-Py track near the 3' SS. We have previously observed that when Prp2 is inactivated using a prp2 temperature-sensitive allele, while most of the pre-mRNA is not processed, some are normally spliced. This observation even more noticeable during meiosis, since pre-mRNA regulation is executed at different levels (splicing, polyadenylation and stability). Using RNAseq, we have determined genome-wide the splicing efficiency of fission yeast cells as they progress into synchronous meiosis, in the presence or absence of functional Prp2. To our surprise, the main characteristic that defines if any given intron requires Prp2 to be spliced depends on the last nucleotides of the preceding exon, next to the 5’ SS in the nucleotide that anneal with U1 RNA. Thus, by simple genetic manipulation, we can modulate the dependency of any given intron on Prp2. We propose that the role of Prp2 consist in the stabilization of a loop between the 5’SS and the 3’ SS, which also depends on the annealing strength between U1 and the 5’ SS.

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2024-10-26 | GSE186973 | GEO

Histone variant H2A.Bbd is associated with active transcription and mRNA processing in human cells

Project description:Variation in chromatin composition and organization often reflects differences in genome function. Histone variants, for example, replace canonical histones to contribute to regulation of numerous nuclear processes including transcription, DNA repair and chromosome segregation. Here we focus on H2A.Bbd, a rapidly evolving variant found in mammals but not in invertebrates. We report that in human cells, nucleosomes bearing H2A.Bbd form unconventional chromatin structures enriched within actively transcribed genes and characterized by shorter DNA protection and nucleosome spacing. Analysis of transcriptional profiles from cells depleted for H2A.Bbd demonstrated widespread changes in gene expression with a net down-regulation of transcription and disruption of normal mRNA splicing patterns. In particular, we observed changes in exon inclusion rates and increased presence of intronic sequences in mRNA products upon H2A.Bbd depletion. Taken together, our results indicate that H2A.Bbd is involved in formation of a specific chromatin structure that facilitates both transcription and initial mRNA processing. This SuperSeries is composed of the following subset Series: GSE38284: Histone variant H2A.Bbd is associated with active transcription and mRNA processing in human cells [ChIP-Seq] GSE38770: Histone variant H2A.Bbd is associated with active transcription and mRNA processing in human cells [RNA-Seq] GSE38890: Histone variant H2A.Bbd is associated with active transcription and mRNA processing in human cells [mRNA] Refer to individual Series

2012-07-15 | E-GEOD-38771 | biostudies-arrayexpress

A CRISPR/Cas9 engineered Von Willebrand factor deficient endothelial cell model shows alternative trafficking of Weibel-Palade body proteins

Project description:Synthesis of the hemostatic protein Von Willebrand factor (VWF) drives formation of endothelial storage organelles called Weibel-Palade bodies (WPBs). In the absence of VWF, angiogenic and inflammatory mediators that are co-stored in WPBs are subject to alternative trafficking routes. In Von Willebrand disease (VWD) patients, the partial or complete absence of VWF/WPBs may lead to additional bleeding complications, such as angiodysplasia. Studies addressing the role of VWF using VWD patient-derived blood outgrowth endothelial cells (BOECs) have reported conflicting results due to the intrinsic heterogeneity of patient-derived BOECs. To study the role of WPBs in endothelial cells using CRISPR-mediated knockout of VWF in BOECs. We used CRISPR/Cas9 gene editing in single donor cord blood-derived BOECs (cbBOECs) to generate clonal VWF-/- cbBOECs. Clones were selected using high-throughput screening, VWF mutations were validated by sequencing and cells were phenotypically characterized. Two VWF-/- BOEC clones were obtained and were entirely devoid of WPBs, while overall cell morphology was unaltered. Several WPB proteins, including CD63, syntaxin-3 and the cargo proteins Ang-2, IL-6 and IL-8 showed alternative trafficking and secretion in absence of VWF. Interestingly, Ang-2 changed localization to the cell periphery and colocalized with Tie-2. CRISPR editing of VWF provides a robust method to create VWF deficient BOECs that can be directly compared to their wild-type counterparts. Results obtained with our model system confirmed alternative trafficking of several WPB proteins in the absence of VWF and support the theory that increased Ang-2/Tie-2 interaction contributes to angiogenic abnormalities in VWD patients.

2019-11-11 | PXD013857 | Pride

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