OC 43.1 - Idntification of a von Willebrand factor homozygous deep intronic variant by next-generation analysis, and subsequent validation by mRNA analysis and study of the patient-derived endothelial cells

Monday, July 11, 2022

2:45 PM – 3:00 PM

Location: ExCel Center, ICC Capital Suite Room 7&12

Presentation Format: In-Person

Presenting Author(s)

HY

Hamideh Yadegari, PhD

Research Group Leader
University Clinic Bonn Institute of Experimental Hematology and Transfusion Medicine Venusberg-Campus 1
Bonn, Nordrhein-Westfalen, Germany

Co-Author(s)

MJ

Muhammad Ahmer Jamil

Dr
Institute of Experimental Haematology and Transfusion Medicine and Center for Rare Diseases, University Hospital Bonn, Bonn, Germany
bonn, Nordrhein-Westfalen, Germany
Johannes Oldenburg

Professor, Chairman and Director
University Clinic Bonn AöR, Institute of Experimental Haematology and Transfusion Medicine, Bonn, Germany
Bonn, Nordrhein-Westfalen, Germany

Background: 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 VWF gene (VWF).

Aims:

Aims: In this study, we intended to elucidate causative mutation through DNA analysis of the entire VWF gene, VWF transcript analysis, and study of the patient-derived endothelial colony-forming cells (ECFCs).

Methods: Whole-genome NGS analysis was executed on an Illumina platform. ECFCs were obtained from the peripheral blood of the IP and healthy donors. Total RNA from ECFCs were isolated, and VWF mRNA was quantified using the TaqMan assay. Additionally, Whole-transcriptome RNA-sequencing was completed on Illumina HiSeq 2500 V4 platform. Amount of VWF antigen (VWF:Ag) secreted into the medium and present in lysates of ECFCs were measured. Immune-staining of the ECFCs was done by using anti-human VWF and Aniopoeitin 2 antibodies. Subsequently, image acquisition was carried out using a Carl Zeiss Apotome.2 microscope.

Results: 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. Illumina artificial intelligence (AI) splicing prediction software, 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, Weible-palade bodies.

Conclusion(s): Our findings underline the potential of NGS in conjunction with RNA analysis, and patient-derived cells studies for genetic diagnosis of mutation-negative type 3 VWD patients.