A Homozygous Deep Intronic Variant Causes Von Willebrand Factor Deficiency and Lack of Endothelial-Specific Secretory Organelles, Weibel–Palade Bodies

Abstract

A type 3 von Willebrand disease (VWD) index patient (IP) remains mutation-negative after completion of the conventional diagnostic analysis, including multiplex ligation-dependent probe amplification and sequencing of the promoter, exons, and flanking intronic regions of the VWF gene (VWF). In this study, we intended to elucidate causative mutation through next-generation sequencing (NGS) of the whole VWF (including complete intronic region), mRNA analysis, and study of the patient-derived endothelial colony-forming cells (ECFCs). The NGS revealed a variant in the intronic region of VWF (997 + 118 T > G in intron 8), for the first time. The bioinformatics assessments (e.g., SpliceAl) predicted this variant creates a new donor splice site (ss), which could outcompete the consensus 5′ donor ss at exon/intron 8. This would lead to an aberrant mRNA that contains a premature stop codon, targeting it to nonsense-mediated mRNA decay. The subsequent quantitative real-time PCR confirmed the virtual absence of VWF mRNA in IP ECFCs. Additionally, the IP ECFCs demonstrated a considerable reduction in VWF secretion (~6% of healthy donors), and they were devoid of endothelial-specific secretory organelles, Weibel–Palade bodies. Our findings underline the potential of NGS in conjunction with RNA analysis and patient-derived cell studies for genetic diagnosis of mutation-negative type 3 VWD patients.

Potentials of Endothelial Colony-Forming Cells: Applications in Hemostasis and Thrombosis Disorders, from Unveiling Disease Pathophysiology to Cell Therapy

“…The data obtained from these studies led to the discovery the novel pathogenesis mechanisms leading to VWF deficiencies in VWD patients and clarifying the effect of VWF variants on intracellular processing of VWF, including multimerization, storage/formation of WPBs, and secretion. 22,[44][45][46][47][48][49][50][51] The use of the patient-derived ECFCs emphasized the significance of the exonic synonymous and deep intronic variants on VWF splicing and their contribution to the pathogenesis of VWD. [44][45][46][47]51 Furthermore, we inspected the gene expression profile and trafficking of the inflammatory/angiogenesis proteins Ang2 and P-selectin, co-stored with VWF in WPBs, in the patient-and healthy control-derived ECFCs.…”

“…22,[44][45][46][47][48][49][50][51] The use of the patient-derived ECFCs emphasized the significance of the exonic synonymous and deep intronic variants on VWF splicing and their contribution to the pathogenesis of VWD. [44][45][46][47]51 Furthermore, we inspected the gene expression profile and trafficking of the inflammatory/angiogenesis proteins Ang2 and P-selectin, co-stored with VWF in WPBs, in the patient-and healthy control-derived ECFCs. Subsequently, we demonstrated alterations in intracellular trafficking of Ang2 and P-selectin and gene expression profiles of patient-derived ECFCs, even in distinct patterns, highlighting the implication of ECFCs as a valuable source in VWD research.…”

“…Subsequently, we demonstrated alterations in intracellular trafficking of Ang2 and P-selectin and gene expression profiles of patient-derived ECFCs, even in distinct patterns, highlighting the implication of ECFCs as a valuable source in VWD research. 45,46 Correspondingly, Schillemans and colleagues reported alternative trafficking and secretion of several WPBs proteins (Ang-2, IL-6, and IL-8) after generating VWF-knockout ECFCs using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) gene-editing method. With this study, they also showed that CRISPR/Cas9 strategy is a robust method to create VWF-deficient/or -defective ECFCs that can be used for investigating pathophysiological mechanisms of VWD.…”