Abstract

von Willebrand disease (VWD) is the most commonly inherited bleeding disorder with a symptomatic prevalence of 1:1000. Type 1 VWD comprises ∼80% of VWD cases and is defined by mild to moderate deficiencies of von Willebrand factor (VWF). Aberrant splicing causes 10-15% of type 1 VWD cases and the mutations are primarily found in consensus splice sites. Investigation of the VWF mRNA is required in order to determine whether the sequence variants outside of consensus splice sites affect splicing. In this study, aberrant splicing of VWF was studied using platelet and/or blood outgrowth endothelial cell (BOEC) mRNA from 3 patients from two type 1 VWD families with putative exonic and intronic splicing mutations. Variant splice forms were identified and their effects were further characterized through expression studies in HEK293(T) cells, as well as further investigation of the patient-derived BOEC. Table 1 displays the patient phenotype, mutation status, and the VWF splice forms identified through reverse transcription of the patient mRNA.

Table 1

Patient phenotype, mutation, and identified splice variants

Patient ID Bleeding Score VWF: Ag (IU/mL) VWF:RCo (IU/mL) FVIII:C (IU/mL) Nucleotide Change VWF Splice Variants 
V70 11 0.27 0.17 0.45 c.3538G>A heterozygous wildtype (wt) VWF exon 23 skipping exon 26 skipping exons 23&26 skipping 
V447 11 0.40 0.38 0.51 c.6599-20A>T heterozygous wt VWF exon 38 skipping 
V449 0.44 0.44 0.75 c.6599-20A>T heterozygous wt VWF exon 38 skipping 
Patient ID Bleeding Score VWF: Ag (IU/mL) VWF:RCo (IU/mL) FVIII:C (IU/mL) Nucleotide Change VWF Splice Variants 
V70 11 0.27 0.17 0.45 c.3538G>A heterozygous wildtype (wt) VWF exon 23 skipping exon 26 skipping exons 23&26 skipping 
V447 11 0.40 0.38 0.51 c.6599-20A>T heterozygous wt VWF exon 38 skipping 
V449 0.44 0.44 0.75 c.6599-20A>T heterozygous wt VWF exon 38 skipping 

The skipping of exon 23, 26, or the combined skipping of these two exons are in-frame changes to the VWF mRNA and therefore truncated VWF would be produced from these transcripts. Skipping exon 38 however, introduces a premature termination codon (PTC) which may induce nonsense mediated decay (NMD) of the VWF mRNA. Site-directed mutagenesis was used to create expression vectors for each of the aberrant VWF splice variants, which were then transfected into HEK293T cells at varying ratios mutant:wt VWF (100:0, 50:50, 25:75, 10:90, 0:100). All 4 of these VWF variants displayed a significant dose dependent impairment of VWF secretion relative to the wt transfections (Table 2). This decrease in VWF secretion was only matched by a corresponding increase in VWF in the cell lysates for the exon 26 skipping transfections (p<0.05) indicating intracellular retention of the VWF. Where intracellular retention was not observed, proteasomal degradation of the mutant VWF was proposed as the pathogenic mechanism. Proteasomes were inhibited by MG-132; however, intracellular VWF did not increase with treatment and therefore an alternate pathway may be responsible for decreased expression of these proteins. Additionally, calcium ionophore did not appear to stimulate secretion of the VWF mutants lacking either exon 26, exons 23&26, or exon 38 in the absence of wt VWF (Table 2).

Table 2

VWF secretion (media/total VWF) relative to wt and fold change (fc) with calcium ionophore stimulus (*** p<0.001; ** p<0.01; *p<0.05)

 Exon 23 Skipping Exon 26 Skipping Exons 23&26 Skipping Exon 38 Skipping 
% mutant vector wt(%) fc wt(%) fc wt(%) fc wt(%) fc 
100 5±3.1 *** 4.7±0.2 7±2.3 *** 0.4±0.4 14±5.1 *** 0±0.02 2±0.6*** 0.96±0.1 
50 29±20* 2.3±0.1* 11±4.5 *** 14.1±2.2 64±12* 2.3±1.7* 58±17* 1.3±0.1 
25 52±21 *  53±15 *  79±41  68±19 *  
10 79±21  80±28  113±52  84±10  
 Exon 23 Skipping Exon 26 Skipping Exons 23&26 Skipping Exon 38 Skipping 
% mutant vector wt(%) fc wt(%) fc wt(%) fc wt(%) fc 
100 5±3.1 *** 4.7±0.2 7±2.3 *** 0.4±0.4 14±5.1 *** 0±0.02 2±0.6*** 0.96±0.1 
50 29±20* 2.3±0.1* 11±4.5 *** 14.1±2.2 64±12* 2.3±1.7* 58±17* 1.3±0.1 
25 52±21 *  53±15 *  79±41  68±19 *  
10 79±21  80±28  113±52  84±10  

Pseudo Weibel-Palade body (WPB) localization of the VWF was observed through confocal immunofluorescence of HEK293 cells transfected with wt VWF and VWF skipping exon 26. VWF skipping exon 23 and exons 23&26 show some localization of the VWF in rounded organelles; however this is less organized than was seen with wt VWF. Staining of VWF lacking exon 38 appears to resemble retention in the ER, and co-localization with PDI will be investigated.

Overall, we have found that the exonic mutation c.3538G>A causes skipping of exons 23 and/or 26 which cause type 1 VWD in the heterozygous patient V70 through the combined action of intracellular retention of VWF lacking exon 26, and decreased secretion of VWF skipping exon 23 and VWF skipping exons 23&26. Additionally, the skipping of exon 38 from the intronic mutation c.6599-20A>T causes type 1 VWD in patients V447 and V449 likely due to NMD of the PTC containing transcript and potentially ER degradation of the protein product of any residual mutant mRNA. Therefore these mutations lead to pathogenic VWF splicing variants which cause type 1 VWD through multiple mechanisms.

Disclosures:

James:CSL Behring: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; Baxter: Honoraria; Bayer: Honoraria.

Author notes

*

Asterisk with author names denotes non-ASH members.