A Large Pedigree with Von Willebrand Disease (VWD) Caused by a Novel Unifying Mutation (M1304R) Exhibits Significant Laboratory and Clinical Phenotypic Heterogeneity.
Authors: Di Paola Jorge , Chen Junmei , Montgomery Robert R. , Haberichter Sandra L. , Lopez Jose A. 
Published: October 2019

Abstract 542
We have characterized a pedigree with VWD that exhibits significant phenotypic variability. The pedigree is composed of 24 Caucasian members and includes 12 individuals diagnosed with VWD, based on abnormal VWF levels and the presence of mucocutaneous bleeding. The pattern of transmission is autosomal dominant. Mutational analysis of exon 28 of the VWF locus was performed in all individuals. A novel single base T>G substitution at position 3911 that predicts a methionine to arginine (M1304R) change in the A1 domain of the mature VWF molecule was detected in all affected individuals, and segregates with the bleeding phenotype. Sequence of the entire coding region of VWF in 1 affected and 1 non-affected member did not show any additional mutations. Von Willebrand factor antigen levels (VWF:Ag) and von Willebrand factor ristocetin cofactor activity (VWF:RCo) in the affected individuals are variable with a mean of 21.9 IU/dL (range 11 to 30) and 11.18 IU/dL (range 3 to 17) respectively. Platelet counts in all individuals of the pedigree are within the normal range. While bleeding scores in non-affected members are below 2 (no bleeding phenotype), they range from 3 to 7 among mutation positive individuals (indicating a wide variation in bleeding symptoms). Multimer analyses also show significant variability, with some individuals having a full range of multimer sizes and others only having low and medium size molecular weight multimers. This loss of high molecular weight multimers does not appear to correlate with increased bleeding tendency. We also studied, by ELISA, plasma from affected and non-affected individuals of the pedigree using a recently developed llama antibody (nanobody AU/VWFa-11) which recognizes 2B and/or 2M mutations of the A1 domain. A ratio (“activation factor”) that represents the relative amount of mutant VWF in patient plasma was generated between patients nanobody-bound VWF and that derived from normal pooled plasma. Nanobody binding in the patients was greater than in the unaffected family members, but varied considerably among affected members, with “activation factors” that range from 1.1 to 4.9. Since all individuals exhibit the same mutation this variation is not likely due to differences in binding affinity of patient VWF to the nanobody nor is it due to the difference in VWF concentration because the ratios are independent of VWF concentration. The variation in “activation factor” suggests that plasma VWF in the affected individuals contains different amount of mutant protein, and this may account for the wide range of bleeding tendencies among these patients. Expression of the M1304R VWF mutant in HEK293 cells show decreased secretion. It also fails to form storage granules, and the VWF secreted lacks high molecular weight multimers. When co expressed with the wild-type allele, total VWF secretion is decreased, multimerization is nearly normalized but still no storage granules are formed in HEK293 cells, suggesting that this particular mutation affects multimerization and trafficking. In summary, we have carefully characterized a family with VWD caused by a unifying mutation that exhibits significant phenotypic variability, which cannot be predicted by current availabel techniques. In order to further understand the clinical and laboratory variability of VWD, it is important to develop new technologies to quantify amounts of mutant VWF in plasma from VWD patients.

Disclosures:
No relevant conflicts of interest to declare.