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

• Published in: International journal of molecular sciences 2022-03, Vol.23 (6), p.3095
• Main Authors: Yadegari, Hamideh, Jamil, Muhammad Ahmer, Marquardt, Natascha, Oldenburg, Johannes
• Format: Article
• Language: English
• Subjects: angiopoietin-2; Antigens; Bioinformatics; Colony-forming cells; deep intronic mutation; Disease prevention; Exons; Flow cytometry; Genetic screening; Glycoproteins; Homozygote; Humans; Introns - genetics; Microscopy; mRNA turnover; Mutation; Neural networks; Next-generation sequencing; Nonsense mutation; Nonsense-mediated mRNA decay; Organelles; Patients; RNA, Messenger - genetics; RNA, Messenger - metabolism; Stop codon; von Willebrand disease; Von Willebrand factor; von Willebrand Factor - genetics; von Willebrand Factor - metabolism; VWF gene; Weibel-Palade Bodies - genetics; Weibel-Palade Bodies - metabolism; Weibel–Palade bodies
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms23063095

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.