Epigenetic Regulation of Von Willebrand Factor Gene Expression May Contribute to Von Willebrand Disease Severity

Background: Von Willebrand disease (VWD) is the most common inherited bleeding disorder with a prevalence ranging from 0.8 to 1.3%. VWD is characterized by incomplete penetrance, variable expressivity with markedly variable VWD antigen levels, even in families with a unifying VWF mutation, suggesting the presence of modifier genes or epigenetic modifications affecting expression of the VWF gene. We had previously diagnosed and characterized an 821-member, multigenerational Amish family in which 121 individuals exhibit a single autosomal-dominant C4120T mutation (R1374C) in the A1 domain of the mature VWF molecule. VWF antigen levels were found to vary significantly, from 9-76 in the individuals exhibiting the C4120T mutation vs 46-483 in the individuals not exhibiting the mutation. To date, very little is known about the epigenetics of VWD and no data exist investigating promoter methylation as a modifier of disease severity. We hypothesized that VWF gene is epigenetically silenced by aberrant DNA methylation resulting in differential expression of VWF protein in patients with VWD. We report our findings suggesting that promoter methylation within the VWF gene may modify VWD severity.

Methods: Genomic DNA previously extracted from whole blood using DNeasy kit was CT converted with the use of the EZ DNA Methylation Kit. Primers encompassing a CpG island containing 4 CpG sites in the 5’ region of the VWF gene spanning from 138 to 490 were designed using MethPrimer. The resulting PCR products were gel-extracted and purified, cloned using the TOPO TA Cloning Kit, transformed into competent, Amp resistant E. coli cells and grown on an Ampicillin containing media. This was done in 30 samples, 20 of which contain the disease-characterizing C4120T mutation and 10 which did not. Approximately 20 colonies from each of the 30 samples were randomly chosen and grown in culture media. Plasmid DNA was extracted and sequenced on an ABI-Prism 3100 Genetic Analyzer. The methylation status of 4 CpG dinucleotides of the VWF promoter regions was analyzed using BiQ Analyzer software.

Results: We performed ANOVA comparing the high VWF antigen level clones against the low VWF antigen level clones within the C4120T mutation subset and similarly for the controls. Methylation percentage was higher in the low antigen level group at each of the four CpG sites although none reached statistical significance. At two sites, we achieved near statistical significance CpG1 (p=0.100) and CpG2 (p=0.063). Among the control individuals, we did not observe this trend. Methylation was higher in the low antigen group vs the high antigen level group at CpG sites 2 and 3, but lower at CpG sites 1 and 4 with CpG4 bordering on statistical significant (p=0.053). We also performed a Spearman Rank-Order Correlation Coefficient which yielded a near-significant trend between VWF antigen level and methylation percentage within the mutation-bearing, low-expression subset at CpG4 (p=0.064).

Discussion: Promoter methylation is known to modify gene expression in numerous disease processes. For the first time, we demonstrate a possible epigenetic effect contributing to the known variance in severity of von Willebrand Disease that is known to correlate with VWF antigen levels. Although our data do not achieve statistical significance, we observed a trend of higher methylation associated with low antigen levels in individuals with the C4120T mutation at two CpG sites. We also observed a near significant association between degree of methylation and VWF antigen levels at 1 CpG site within the mutation-bearing, low antigen level subset. Further studies with larger number of individuals will confirm or refute this observation.