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Pathogenicity assessment of genetic variants in von Willebrand disease using quantitative, qualitative, and functional approaches



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Sims, Matthew 


Von Willebrand disease (VWD) is the most common inherited bleeding disorder. It is defined by a deficiency or dysfunction of plasma von Willebrand factor (VWF), a glycoprotein with a multifaceted role in haemostasis. The majority of circulating VWF is synthesised and released by endothelial cells (ECs). VWD is caused by rare DNA sequence variants in the VWF gene. However, coupling genotype with phenotype is complicated by factors including incomplete penetrance and the trans-acting effect of the ABO histo-group. High throughput sequencing (HTS) is becoming the standard of care for the diagnosis of inherited bleeding disorders, including VWD. This raises several challenges. First, how should candidate VWF variants be searched for and their pathogenicity assessed? Second, if a pathogenic variant (PV) for VWD is identified how does this influence bleeding risk? Third, if the mechanism of the identified PV is unknown, how can its effect be elucidated? These questions are sequentially addressed in this thesis. I curated 1,455 unique VWF variants into a single repository called VWDbase. Variants were only included if they had been previously linked to VWD. Two thirds of VWDbase variants had previously been deemed causal of VWD and were termed Putatively Aetiological VWD Variants (PAVVs). Of these, 194 PAVVs were identified in the whole exome sequencing data of 140,327 participants in UK Biobank (UKB). These data were used to accurately determine the minor allele frequency (MAF) of these PAVVs. The pathogenicity of each PAVV was then scrutinised using published data. Seventy three of 194 PAVVs were rejected as being pathogenic for VWD. In over half of cases this was because the PAVV occurred too frequently to be compatible with VWD prevalence. The PAVVs that were accepted as being pathogenic for VWD were identified in 401 UKB participants (the ‘genetically accepted VWD’ [ga] group). Hospital inpatient data were analysed for UKB participants from 1997 to 2020. These were used to create the ICD-bleeding assessment tool (ICD-BAT) to assess the presence or absence of bleeding episodes across 16 different domains and the time over which UKB participants lived without experiencing an episode (bleeding free survival). There was no difference in the ICD-BAT score or bleeding-free survival when the gaVWD group was compared to the rest of the UKB population. However, blood group O predicted for both an increased ICD-BAT score and a reduced risk of bleeding-free survival over the observation period. VWDbase was then utilised to analyse 10 patients with VWD in whom no molecular diagnosis had previously been identified. The patient with the most severe (type 3) VWD phenotype was homozygous for a rare PAVV, c.8155+6T>A, situated in the donor splice site of the penultimate exon-intron junction. Analysis of platelet mRNA demonstrated that c.8155+6T>A results in a transcript with a frameshift and premature termination codon (PTC). Evaluation of patient-derived endothelial colony forming cells (ECFCs) revealed that c.8155+6T>A resulted in VWF that was mostly retained in a perinuclear position as opposed to being packed into Weibel-Palade bodies (WPBs). In order to overcome the finite supply of ECFCs and assess the effect of c.8155+6T>A in a different genetic context, a new cellular model of VWD was created. Human induced pluripotent stem cells (hiPSCs) were edited using CRISPR/Cas9 to contain a PTC in exon 50, positioned 10 nucleotides 5’ of c.8155+6T>A. They were then differentiated to ECs and the findings in the patient ECFCs were replicated. The effect of c.8155+6T>A is likely to be due to the truncation of VWF prior to the C-terminal cysteine knot (CK), the domain which is crucial for VWF dimerisation and exit from the endoplasmic reticulum. In summary, this thesis highlights the utility of large reference populations and hiPSC-derived ECs (iECs) in the critical appraisal of PAVVs.





Ouwehand, Willem
Frontini, Mattia


von Willeband disease, haematology, genetics, genomics, bleeding, coagulation, von Willebrand factor, endothelial cells, induced pluripotent stem cells, CRISPR/Cas9, c.8155+6T>A


Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge
Medical Research Council Clinical Research Training Fellowship (grant reference MR/R002363/1)
Is supplemented by: