Introduction: In recent years, the role of copy number variation (CNV) in the pathogenesis of type 1 von Willebrand disease (VWD) has been extensively explored. Large cohort studies including the MCMDM-1VWD and French studies have identified a total of eight partial VWF gene deletions, involving single and multiple exons, as well as one duplication. Furthermore, breakpoints have been identified within the MCMDM-1VWD cohort study for deletions involving exons 3, 32-34 and 33-34.
Aim: To characterize and determine heterozygous deletion breakpoints of a deletion within the Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB-VWD) cohort, previously identified through array CGH analysis and to determine the pathogenesis of the deletion through in vitro analysis.
Methods: Identification of deletion breakpoints utilized a deletion-specific multiplex PCR approach, whereby two forward primers and one reverse primer were designed to determine the heterozygous exon deletion, based on previous optimization within the MCMDM-1VWD cohort. VWF antigen (VWF:Ag), VWF propeptide (VWFpp), and multimer distribution were performed in a central laboratory. Recombinant exon 33-34 deletion mutant (rVWFdel33-34) was created using site directed mutagenesis. Recombinant vectors were transiently transfected into HEK293T cells and Renilla used as a transfection control. Conditioned media and cellular lysates were collected 48hr post-transfection and analysed by VWF:Ag ELISA, comparing to wild-type VWF (rVWFwt) expression.
Results: Three families enrolled in the ZPMCB-VWD displayed a deletion of VWF exon 33-34. The deletion was determined to be 3.4kb in size and led to an in-frame loss of exons 33-34, with breakpoints being identical to those identified in the MCMDM-1VWD study (c.5620+872_5842+2440delinsGCAGCATAAGCATAAAGC). VWFpp/VWF:Ag ratios were elevated in all affected family members with the exon 33-34 deletion (mean 4.3) as opposed to unaffected family members (mean 1.3). These results are consistent with findings in the MCMDM-1VWD family (mean VWFpp/VWF:Ag of 4.8), suggesting increased clearance of VWF. In silico analysis of the breakpoint region suggested no significant repetitive elements and microhomology mediated end-joining was hypothesised as the potential mechanism leading to the deletion. Expression of recombinant mutant rVWFdel33-34 revealed that secretion reduced by 52% in the homozygous state (p<0.0001) when compared to rVWFwt expression. The deletion also resulted in a significant 35% reduction in secretion in the heterozygous state (p<0.0001). However, no intracellular retention was observed in either mutant state.
Conclusion: Analysis of the exon 33-34 deletion breakpoint revealed no highly homologous repetitive sequences but there was a DNA insertion of 18 bp at the deletion breakpoint. In vitro expression of the exon 33-34 deletion suggests that there is a moderate effect on VWF secretion into conditioned media, without increased intracellular retention. The exon 33-34 deletion leads to the loss of nine cysteine residues. Inter- and intra- disulphide bonding between cysteine residues within VWF monomers and multimers is known to be functionally important in facilitating correct VWF structural orientation and folding within D4-CK domains. Loss of these cysteines may explain the moderate reduction in VWF secretion and potentially reduced plasma survival of the variant VWF.
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