The dominant p.Thr274Pro mutation in the von Willebrand factor propeptide causes the von Willebrand disease type 1 phenotype in two unrelated patients.

BACKGROUND:von Willebrand factor propeptide (VWFpp) plays an important role in VWF multimerization and storage. VWFpp mutations have been previously associated with types 1, 3 and 2A/IIC von Willebrand disease (VWD). AIMS:To characterize the novel p.Thr274Pro variant identified in two unrelated type 1 VWD patients. METHODS:Phenotype tests were performed to evaluate patients' plasma and platelets following the current ISTH-SSC guidelines. Molecular analysis was performed using next-generation sequencing. The pcDNA3.1-VWF-WT and mutant pcDNA3.1-VWF-Thr274Pro expression vectors were transiently transfected into HEK293 cells to evaluate recombinant (r)VWF constitutive and regulated secretion. For the latter, the transfected cells were stimulated with phorbol-12-myristate-13-acetate. Immunofluorescence staining was performed to assess the localization of WT-rVWF and Thr274Pro-rVWF in endoplasmic reticulum, lysosomes, cis-/trans-Golgi and pseudo-Weibel Palade bodies. RESULTS:Biochemical characterization of patients' plasma samples indicated a type 1 VWD diagnosis. Both patients were heterozygous for the p.Thr274Pro variant. Hybrid Thr274Pro/WT-rVWF showed a secretion reduction of 36±4% according to patients' plasma VWF:Ag levels, whereas Thr274Pro-rVWF secretion was strongly impaired (21±2%). The amount of rVWF in cell lysates was nearly normal for both Thr274P (62±17%) and Thr274Pro/WT-rVWF (72±23%). The regulated secretion was impaired for Thr274Pro/WT-rVWF, whereas Thr274Pro-rVWF was not released at all. Immunofluorescence staining revealed no particular differences between WT and Thr274Pro-rVWF, although Thr274Pro-rVWF showed less pseudo-Weibel Palade bodies with a rounder shape than WT-rVWF. CONCLUSIONS:The novel p.Thr274Pro mutation has a dominant effect and it is responsible of patients' type 1 VWD phenotype through a combined mechanism of reduced synthesis, impaired secretion and multimerization.