An Association Study of Candidate Modifier Genes in a Large Pedigree with Von Willebrand Disease.
Authors: Di Paola Jorge , Wagner Kayelyn , Nixon Brie , Kline Jacob , Shapiro Amy , Abbott Diana , Burns Trudy , Wang Kai 
Published: April 2020

Von Willebrand Disease (VWD) is a common and highly variable bleeding disorder with incomplete penetrance and variable expressivity. Due to the complexity of VWD it is highly likely that several modifier genes influence its phenotype. We have identified and characterized an extensive Amish pedigree which is composed of 854 individuals of whom 395 have provided blood samples. 71 pedigree members are heterozygous for a missense mutation at position 4120, represented by a single base substitution (C&gt;T) that predicts an arginine to cysteine change at position 1374 (R1374C) in the A1 domain of the mature VWF molecule. Phenotypic characterization of the pedigree included several laboratory measures and a clinical bleeding phenotype determined by a validated bleeding score. The bleeding score was established by a questionnaire that consists of 10 questions that focus on seven areas related to spontaneous and trauma-related mucocutaneous bleeding. The lowest possible score is 0 and the highest is 7. A bleeding score of 3 or higher exhibited a specificity of 0.99 and sensitivity of 0.90 for VWD when given to 97 healthy controls and 63 unrelated individuals diagnosed with VWD. We performed association studies with candidate genes in the pedigree and correlated genotype and allele frequencies with severity of bleeding. The list of candidate variants studied included a total of 27 genetic polymorphisms in 20 genes that encode for the platelet receptors ITGA2, GP1BA, ITGB3 and GP6, for the coagulation factors F2, F5, F7, and F13 and for critical proteins involved in normal hemostasis, thrombosis, fibrinolysis and inflammation such as THBD, SELP, SELE, FGA, FGB, MTHFR, PLAT, PECAM1, CPB2, NOS3, SERPINE and CD14. These gene variants were selected based on their biological significance as well as their minor allele frequency. The differences in genotype distributions and allele frequencies between affected and unaffected pedigree members were evaluated using a proportional odds model for the association analysis. This model, which is a generalization of the usual logistic regression model for a binary outcome, can accommodate a multi-level ordered response variable such as the bleeding score. Five alleles were associated with increased severity of bleeding: Allele C at position 2361 of the SELP gene that encodes for P selectin and has been associated with decreased risk of thrombosis (p= 0.09 and p=0.003 and 0.001 when corrected for mutation status and sex, respectively). Allele C, at position 807 of ITGA2 that has been associated with increased bleeding (p= 0.01, and p= 0.002 and 0.01 when adjusted for mutation status and sex, respectively). Allele G, at position - 260 of CD14 that has been associated with decreased risk for myocardial infarction (p= 0.003, and p = 0.02 and 0.003 when adjusted for mutation status and sex, respectively). Allele T at position 13254 of the GP6 gene that has been associated with bleeding and thrombosis (p= 0.009, and p= 0.05 and 0.01 when adjusted for mutation status and sex, respectively). Finally, allele C at position 677 that has been described as protective for thrombosis (p= 0.02, and p= 0.01 and 0.018 when adjusted for mutation status and sex, respectively). In summary, our results demonstrate that specific genetic variants modify the bleeding phenotype of this pedigree and may contribute to the clinical variability observed in unrelated patients with VWD.