Investigating the pathomolecular mechanisms an in-frame heterozygous von Willebrand factor large deletion in a von Willebrand disease patient with significant bleeding history
H. Yadegari1, N. Marquardt1, V. Ivaskevicius1, O. Rawley2, L. Swystun2, D. Lillicrap2, J. Oldenburg1 (1Bonn, Germany, 2Kingston, Canada)
Bleeding disorders - Basic science
Date: 17.02.2017,
Time: 08:00 - 09:15
Objective: This study aimed to elucidate disease pathogenesis and genetics etiology in an index patient (IP) diagnosed to have type 3 von Willebrand disease (VWD). The IP suffers frequently from bleeding episodes in spite of prophylaxis treatment. The genetic investigations revealed only a de novo heterozygous large deletion of exons 4-34 of von Willebrand (VWF) gene. However, this single defect could not explain low VWF antigen (VWF:Ag) levels and severe clinical demonstration of the IP. The current ex vivo study investigated the molecular pathological mechanisms by which the detected large deletion impairs VWF biosynthesis.
Methods: Blood outgrowth endothelial cells (BOECs) were isolated from peripheral blood of IP and healthy individuals. RNA was extracted from cultured BOECs, and coding region of VWF gene was amplified by RT-PCR. Seventy-two hours after seeding the BOECs, VWF:Ag levels in the medium and lysate of the BOECs were measured. Multimer analysis of the BOECs supernatant was performed. The subcellular location of VWF in BOECs was visualized by immunofluorescence antibody staining and subsequent confocal microscopy scanning.
Results: The BOECs RNA analysis assured biosynthesis of normal VWF transcription originating from wild type allele as well as an aberrant transcript with exons 4-34 deletion. Surprisingly, the mean of secreted VWF:Ag levels from the patient-derived BOECs was only slightly reduced compared with those of the healthy donors (63% vs. 72% respectively). Multimer analysis of the secreted VWF showed loss of large and intermediate multimers along with shift in mobility of low molecular weight multimers. Additionally, confocal immunofluorescent analysis showed a relatively strong VWF staining in IP-derived BOECs. However, it seems that the Weibel-Palade-bodies in IP-BOECs are smaller compared to those within normal BOECs.
Conclusion: In conclusion, our results suggested that production of the VWF from second allele might not be disrupted. Rather it is speculated that in-frame deleted VWF (p.Asp75_Cys1948del) which lacks multimerization domains has a dominant-negative impact on multimerization process. Moreover, we presumed that the secreted chimeric VWF is cleared from the plasma circulate more rapidly than normal VWF. This assumption is remained to be confirmed by mouse model tools.