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Title: Understanding the pathogenesis of copy number variant formation in the von Willebrand factor gene
Author: Cartwright, Ashley
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2013
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von Willebrand disease (VWD) is the most prevalent inherited mucocutaneous bleeding disorder in humans, and results from either quantitative or qualitative defects in circulating levels of the multimeric plasma glycoprotein, von Willebrand factor (VWF). Type 1 VWD, a partial quantitative deficiency of VWF, occurs in 50-70% of affected individuals. The genetic mechanisms involved in the pathogenesis of type 1 VWD were investigated in a European Union cohort study, with no candidate mutation identified in 30% of 150 index cases recruited. A proportion of these patients may be heterozygous for exon deletions of VWF. However, standard DNA sequencing methods undertaken in the original study can mask the presence of copy number variation (CNV) within VWF due to the presence of a wild-type allele. Recent validation of the P011-A1/P012-A1 (MLPA) VWF probe kit enabled analysis of VWF for the presence of heterozygous deletions or duplications (CNV). CNV analysis in this study led to the identification of two novel heterozygous in-frame deletions, which, along with previous deletion identification during optimisation identified 6 deletions in total. This suggested that heterozygous deletions are a significant contributor to the pathogenesis of type 1 VWD. Furthermore, identification, characterisation and in silico analysis of the deletions was undertaken to map the intronic breakpoint locations and understand the pathogenetic mechanisms of deletion formation in VWF. Additional analysis on historical VWF deletions was also undertaken and indicated that VWF was enriched for Alu repetitive elements and these repetitive element sequences are the predominant pathogenetic mechanism in generation of large deletions in VWF. In vitro expression analysis of the in-frame deletion mutants revealed a significant secretion defect of VWF into conditioned media. Furthermore, in some instances, in-frame deletions have a dominant-negative mechanism on VWF secretion, multimerisation and intracellular storage. The phenotypes of patients identified with the heterozygous deletions were found to correlate with the in vitro expression results for all deletions expressed. This provided novel insight into the effects of heterozygous deletions in type 1 VWD.
Supervisor: Goodeve, Anne ; Peake, Ian Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available