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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.

2021-11-30 | GSE161715 | GEO

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

Project description:The term placenta is a highly vascularized tissue and is usually discarded upon birth. Our objective was to isolate clinically relevant quantities of fetal endothelial colony-forming cells (ECFCs) from human term placenta and to compare them to the well-established donor-matched umbilical cord blood (UCB)-derived ECFCs. A sorting strategy was devised to enrich for CD45-CD34+CD31Lo cells prior to primary plating to obtain pure placental ECFCs (PL-ECFCs) upon culture. UCB-ECFCs were derived using a well-described assay. PL-ECFCs were fetal in origin and expressed the same cell surface markers as UCB-ECFCs. Most importantly, a single term placenta could yield as many ECFCs as 27 UCB donors. PL-ECFCs and UCB-ECFCs had similar in vitro and in vivo vessel forming capacities and restored mouse hind limb ischemia in similar proportions. Gene expression profiles were only minimally divergent between PL-ECFCs and UCB-ECFCs, probably reflecting a vascular source versus a circulating source. Finally, PL-ECFCs and UCB-ECFCs displayed similar hierarchies between high and low proliferative colonies. We report a robust strategy to isolate ECFCs from human term placentas based on their cell surface expression. This yielded much larger quantities of ECFCs than UCB, but the cells were comparable in immunophenotype, gene expression, and in vivo functional ability. We conclude that PL-ECFCs have significant bio-banking and clinical translatability potential. Data has been integrated into the Stemformatics web resource http://www.stemformatics.org/datasets/view/6306

2015-03-02 | E-MTAB-3277 | biostudies-arrayexpress

Affymetrix exon array analysis of control and hnRNP H knockdown in 293T cells

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U2AF65Prp2 promotes intron definition by stabilizing U1 snRNP at the 5’ splice site

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.

2021-10-01 | GSE97203 | GEO

Intronic tRNAs of mitochondrial origin regulate constitutive and alternative splicing

Project description:Background: The presence of nuclear mitochondrial DNA (numtDNA) has been reported within several nuclear genomes. Next to mitochondrial protein coding genes, numtDNA sequences also encode for mitochondrial tRNA genes. However, the biological roles of numtDNA, remain elusive. Results: Employing in silico analysis we identify 281 mitochondrial tRNA homologs in the human genome, which we term nimtRNAs (nuclear intronic mitochondrial-derived tRNAs), being contained within introns of 76 nuclear host genes. Despite base changes in nimtRNAs when compared to their mtRNA homologs, a canonical tRNA cloverleaf structure is maintained. To address potential functions of intronic nimtRNAs, we insert them into introns of constitutive and alternative splicing reporters and demonstrate that nimtRNAs promote pre-mRNA splicing, dependent on number and positioning of nimtRNA genes and splice site recognition efficiency. A mutational analysis reveals that the nimtRNA cloverleaf structure is required for the observed splicing increase. Utilizing a CRISPR/Cas9 approach we show that a partial deletion of a single endogenous nimtRNALys within intron 28 of the PPFIBP1 gene decreases inclusion of the downstream located exon 29 of the PPFIBP1 mRNA. By employing a pull-down approach followed by mass spectrometry, a 3’-splice site associated protein network is identified, including KHDRBS1, which we show directly interacts with nimtRNATyr by an electrophoretic mobility shift assay. Conclusions: We propose that nimtRNAs, along with associated protein factors, can act as a novel class of intronic splicing regulatory elements in the human genome by participating in the regulation of splicing.

2020-10-28 | PXD022204 | Pride

Deep intronic homozygous variation in PSMC3, a subunit of the 19S proteasome regulatory complex, causes a syndromic neurosensory disorder combining deafness and cataract

Project description:The association of congenital deafness and early-onset cataracts inherited as a recessive trait is a rare combination described in only a few syndromes with very few genes identified to date. Whole-genome sequencing was performed on 3 patients from independent sibships from a large consanguineous family presenting with severe deafness and early-onset cataracts as part of a variable neurological, sensorial and cutaneous syndrome. Medical assessments and imaging were used to define the phenotype. Genome sequencing was performed to unravel the altered genotype by subsequent bioinformatics analysis based on a specific physiopathological filtering approach. We identified a unique homozygous variant in intron 10 of the PSMC3 gene, encoding the 26S proteasome ATPase ring subunit 5 (Rpt5), with a predicted local splice effect as a new donor site (c.1127+337A>G, p.Ser376Argfs15*) confirmed by RT-PCR. Strikingly, fibroblasts derived from patients carrying the deep intronic homozygous PSMC3 pathogenic variant exhibited strong signs of perturbed protein homeostasis, as evidenced by increased accumulation of intracellular ubiquitin-modified proteins. Most interestingly and in contrast to control cells, patient fibroblasts failed to increase their amount of proteasomes following proteasome inhibition using the TCF11/Nrf1 pathway, indicating that these cells were unable to adapt to proteotoxic stress. By way of 2 different zebrafish assays we further show that loss of function of PSMC3 leads to inner ear development anomalies as well as cataracts alike the patient phenotype. We propose that the PSMC3 proteasome subunit dysfunction leads to a novel human syndrome that includes early onset cataracts and deafness and suggest that Rpt5 plays a major role in inner ear and lens development.

2020-04-28 | PXD015836 | Pride

RegSNPs-intron: a computational framework for predicting pathogenic impact of intronic single nucleotide variants

Project description:Single nucleotide variants (SNVs) in intronic regions have yet to be systematically investigated for their disease-causing potential. Using known pathogenic and neutral intronic SNVs (iSNVs) as training data, we develop the RegSNPs-intron algorithm based on a random forest classifier that integrates RNA splicing, protein structure and evolutionary conservation features. RegSNPs-intron shows excellent performance in evaluating the pathogenic impacts of iSNVs. Using a high-throughput functional reporter assay called ASSET-seq (ASsay for Splicing using ExonTrap and sequencing), we evaluate the impact of regSNPs-intron predictions on splicing outcome. Together, RegSNPs-intron and ASSET-seq enable effective prioritization of iSNVs for disease pathogenesis.

2019-09-30 | GSE138130 | GEO

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Project description:mRNA recovered upon RNF219 IP. [RNA-IP]

| PRJNA377138 | ENA

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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