Genetic Determinants of Plasma Von Willebrand Factor (VWF) and VWF Propeptide Levels

Abstract 3313

Plasma VWF levels vary by approximately five-fold in healthy populations and are influenced by both environmental and inherited factors. Low levels of VWF are associated with bleeding and elevated levels with increased risk for thrombosis. Increased levels of VWF occur with age, may rise acutely due to inflammation or infection, and may serve as a biomarker for endothelial dysfunction and atherosclerosis. Genetic factors are thought to account for 60 – 70% of the variance in VWF antigen levels with ∼30% of this effect due to ABO blood type. VWF multimers are assembled in the Golgi apparatus where the VWF propeptide is cleaved to form mature VWF. Because the half life of plasma VWF propeptide (ppVWF) is ∼5X faster than the mature VWF antigen, the ratio of ppVWF/VWF:Ag in plasma primarily reflects clearance rates of mature VWF while alterations in biosynthesis and/or secretion of VWF affect the level of both ppVWF and VWF:Ag. In order to identify additional quantitative trait loci (QTL) for VWF antigen variation and altered clearance of VWF:Ag, we performed genetic analyses in a healthy young sibling cohort, the Genes and Blood Clotting Study (GABC, n=1,152). The cohort was SNP genotyped with an Illumina Omni-1 array resulting in 763,195 SNPs available for analysis after extensive QC. Plasma VWF antigen (VWF:Ag) and VWF propeptide levels were determined by AlphaLISA (Perkin Elmer, Waltham, MA), using polyclonal anti-human VWF antibodies (DAKO, Glostrup, Denmark) or monoclonal anti-ppVWF antibodies (Blood Center of Wisconsin, Milwaukee, WI). The median levels were 108.1 IU/dL, 99.97 IU/dL and 1.0 for VWF:Ag, ppVWF and ppVWF/VWF:Ag respectively. The estimated heritability (h²) was 64.5%, 55.72%, and 64.26% for VWF:Ag, ppVWF, and ppVWF/VWF:Ag respectively. A genome-wide association study (GWAS) for VWF:Ag in this cohort revealed significant signals at the ABO and VWF loci, consistent with previously published reports. However, GWAS for ppVWF levels demonstrated a signal only at 12p13, overlying the VWF gene itself. The top SNP, rs1800378 (beta 4.297, p-value 1.58E–11), encodes a nonsynonymous substitution (His484Arg) in the VWF propeptide. In contrast, GWAS for ppVWF/VWF:Ag showed a strong signal at 9q34, the ABO locus, (top SNP rs687289, beta −0.2131, p-value 1.88E–62) and no significant signal at 12p13. These results suggest that ABO is the major common determinant of variability in VWF clearance rates and a common SNP, rs1800378, is associated with VWF propeptide levels. We next utilized the sibling structure in this cohort to perform linkage analyses. Linkage analysis for VWF:Ag identified a novel locus at 2p12-2q13 in addition to the expected signal at ABO. The effect size of this chromosome 2 locus on VWF variation (19.2% variance explained) was comparable to the effect of the ABO locus (24.5%). Linkage analysis for ppVWF demonstrated novel loci at 7q21 (LOD score 5.03) and at 11q21 (LOD score 4.12), with no significant signal on chromosome 2. In contrast, linkage analysis for ppVWF/VWF:Ag identified signals at 2p12 (LOD score 3.17), 8p22 (LOD score 3.48) and 11q22 (LOD score 3.41). Taken together, these results identify a number of potential candidate loci for genetic regulation of plasma VWF through alteration of synthesis, storage, secretion or clearance mechanisms. We hypothesize that signals present in linkage but not association are due to VWF regulatory genes with high allelic heterogeneity, harboring many genetic variants that are individually rare, but in aggregate present in a large portion of sibships. Segregation of these variants among sibs could then explain the strong signal by linkage, but lack of signal by association in unrelated individuals. Replication of the strong 2p12-2q13 signal in both VWF:Ag and ppVWF/VWF:Ag linkage studies suggests that the responsible variants at this locus affect VWF levels through altered clearance. The novel QTLs for ppVWF/VWF:Ag levels on chromosome 8p22 and 11q22 are also likely to work through altered clearance mechanisms while the novel signals for ppVWF levels on 7q21 and 11q21, not significant in the VWF:Ag linkage study, may affect efficiency of cleavage or clearance of ppVWF. Identification of the underlying genes at these novel loci and their mechanism of action could lead to improved understanding of the severity, penetrance and treatment for von Willebrand Disease as well as the risk for venous thromboembolic disease associated with elevated VWF.