PB0823 - The rare VWF non-canonical splice-site variant, c.8155+6T>A, causes type 3 von Willebrand disease in homozygosity and results in perinuclear retention of VWF in endothelial cells

Monday, July 11, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Presenting Author(s)

MS

Matthew C. Sims, BA, BMBCh, MRCP, FRCPath

Clinical Research Training Fellow
University of Cambridge
Cambridge, England, United Kingdom

Co-Author(s)

FB

Frances S. Burden

Research Associate
University of Cambridge
Cambridge, England, United Kingdom
JC

Janine H. Collins

Clinical Research Training Fellow
University of Cambridge
Cambridge, England, United Kingdom
PN

Phillip North

Graduate Research Assistant
University of Exeter
Exeter, England, United Kingdom
LS

Luca Stefanucci

Postdoctoral Scientist
University of Cambridge
Cambridge, England, United Kingdom
AD

Adam Dinan

Research Associate
University of Cambridge
Cambridge, England, United Kingdom
SS

Suthesh Sivapalaratnam

Consultant in Haemostasis and Thrombosis
Barts NHS Trust
London, England, United Kingdom
Dan P. Hart, BSc, MBChB, MRCP, FRCPath

Senior Clinical Lecturer in Haematology & Honorary Consultant in Haematology
The Royal London Hospital Haemophilia Centre, Barts and The London School of Medicine and Dentistry
London, England, United Kingdom
Michael A. Laffan, MD

Professor
Centre for Haematology, Imperial College London, London, UK
London, England, United Kingdom
WO

Willem H. Ouwehand

Group Coordinator and Principal Investigator
University of Cambridge
Cambridge, England, United Kingdom
Susan Shapiro, MD, PhD

Consultant Hematologist
Oxford University Hospitals National Health Service Foundation Trust, Oxford, UK
Oxford, England, United Kingdom
MF

Mattia Frontini

Associate Professor
University of Exeter
Exeter, England, United Kingdom

Background: A patient with type 3 von Willebrand disease (VWD3) was enrolled into a whole genome sequencing study as routine genetic analysis had not identified a causal genetic variant. The homozygous single nucleotide variant (SNV), c.8155+6T>A, 4 nucleotides from the intron 51 donor splice site of VWF, was suspected to be aetiological.

Aims: To assess the functional consequences of c.8155+6T>A.

Methods: The proband and controls were consented for studies approved by the East of England research ethics committee. Blood was taken from which platelet cDNA and endothelial colony forming cells (ECFCs) were derived. Subsequently, VWF-directed CRISPR-Cas9 RNAs were transduced into wild type (WT) human induced pluripotent stem cells (hiPSCs). Two clones were selected with different biallelic termination codons: at 120 nucleotides 3' from the translation start site (knock out, KO); and 12 nucleotides 5’ of the position of the proband’s SNV (Δexon50-52). WT, KO, and Δexon50-52 hiPSCs were differentiated to endothelial cells (iECs).

Results: c.8155+6T>A resulted in the skipping of exon 50 (Figs. 1A and 1B), and consequently a frameshift and termination codon 25 amino acids 3’ of exon 49 (Fig. 1C). In the proband ECFCs, VWF was predominantly distributed in a perinuclear halo with minimal packaging into punctate structures (Fig. 1D). These findings were replicated in iECs (Fig. 2). Furthermore, 239 iECs were evaluated: 71% of Δexon50-52 iECs did not contain VWF compared with 9% of WT (P = 2.1 x 10-14, Fisher Exact Test). Most of the remaining 29% of Δexon50-52 iECs contained VWF in a perinuclear distribution.

Conclusion(s): Homozygous c.8155+6T>A results in VWD3 due to a reduction in abundance and packaging of VWF in endothelial cells. This is likely to be due to truncation of VWF prior to the C-terminal cysteine knot, which is crucial for VWF dimerisation and exit from the endoplasmic reticulum.