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Title: Copy number variation in von Willebrand disease : screening, prevalence and functional characterisation
Author: Webster, Simon
ISNI:       0000 0004 6056 6903
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2016
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von Willebrand disease (VWD) is the most common mucocutaneous bleeding disorder in humans. It is caused by genetic defects/disruptions in the von Willebrand factor gene (VWF) locus. VWF encodes a large multimeric glycoprotein (VWF), essential for platelet dependent primary haemostasis and binding, and transportation of coagulation factor VIII. VWF is stored in specialised storage organelles called Weibel-Palade bodies (WPB). Disruption of VWF may result in qualitatively defective (type 2), or quantitatively deficient (types 1 and 3) VWD. It has been reported that in type 1 VWD, ~35% of patients have no causative VWF mutation. Copy number variation (CNV) consisting of large exonic deletions and/or duplications within VWF have been reported. However, these analyses were limited to exons. The initial aim of this thesis was to design a custom VWF microarray to enable comprehensive array comparative genomic hybridisation (aCGH) based CNV screening across the entire VWF locus including exonic, intronic and 5' and 3' flanking intergenic regions. Custom aCGH CNV analyses identified a range of exonic CNV in both type 1 and type 3 VWD patients. In addition two novel 5' intergenic deletions were identified ~9 Kb and 22 Kb upstream of the ATG initiator site. In silico analysis of these deletions suggested that they possess active regulatory elements and may be involved in the regulation of VWF gene expression. A multiplex genotyping assay was developed to enable high-throughput screening of these deletions in the population. The pathogenic contribution of VWF exonic CNV to quantitative VWD is poorly understood. In this study in vitro analysis of three VWF exonic deletions, involving exons 3, 32-34 and 33-34 demonstrated defects in VWF secretion and biosynthesis. These findings were supported by high resolution, widefield fluorescent, and super resolution, structured illumination microscopy (SIM). These data revealed profound defects on VWF storage and WPB biogenesis.
Supervisor: Goodeve, Anne ; Hampshire, Dan Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available