No abstract

Missense mutations are not considered a common cause of type 3 von Willebrand's disease (VWD), the most severe defect of von Willebrand factor (VWF) characterized by undetectable levels of this protein in plasma and platelets. Nevertheless, several missense mutations have been identified in these patients. In this study, we report the cases of two Italian patients with type 3 VWD, both compound heterozygotes for different missense mutations and null alleles, p.D141Y/c.2016_2019del and p.C275S/p.W222X. We performed in vitro expression studies of the candidate missense mutations, both located in the D1 domain of VWF propeptide, to confirm their link with the disease and to understand the mechanisms of type 3 VWD responsible in these patients. Mutant and wild-type (WT) expression vectors were used for transient transfection and co-transfection studies in COS-7 cells. Single construct transfections of both missense mutations showed a strongly reduced but detectable secretion of recombinant (r)VWFs (approximately 15% of WT), with essentially only dimers being visualized on multimeric analysis. As expected, expression of a single construct of either mutation with the WT, showed mildly reduced secretion (approximately 40% of WT) and a full set of multimers. These expression studies indicate that the two amino acids D141 and C275 are key residues in the tertiary structure of the VWF propeptide. Their replacement with a tyrosine and a serine, respectively, might compromise propeptide folding, affecting both its intracellular survival and its capacity to mediate multimerization. Co-expression of hybrid rVWFs confirmed the recessive inheritance pattern of these missense mutations.

Summary.  Background: Type IIH von Willebrand disease was reported 20 years ago as a novel variant characterized by the loss of the largest multimers in plasma and platelets and absence of the typical triplet structure. Objectives and methods: The propositus and his daughter have been reinvestigated and characterized at the molecular level. The identified mutations were expressed in COS‐7 cells to evaluate the mechanism of this variant. Results and Discussion: The propositus had normal von Willebrand factor (VWF):ristocetin cofactor activity (RCo) and high VWF antigen (VWF:Ag) values, with a low VWF:RCo/VWF:Ag ratio (0.51). No abnormalities were found in his daughter, except for the reduced triplet structure in plasma VWF and diminished ultralarge VWF (ULVWF) multimers in platelets. Three mutations were identified in the propositus: 604C>T (R202W), 4748G>A (R1583Q), and 2546G>A (C849Y). The amounts of secreted recombinant VWF (rVWF) were apparently increased for R202W (130%), R202W‐R1583Q (131%), and R202W‐R1583Q/WT (121%), reduced for C849Y (72%) and C849Y/WT (83%), and normal for R1583Q (107%) and R202W‐R1583Q/C849Y (102%). In cell lysates, higher values were found in association with the C849Y mutation. A normal multimeric pattern was found in R1583Q rVWF, mainly dimers in R202W rVWF, and intermediate molecular weight multimers in C849Y rVWF. Hybrid R202W‐R1583Q/WT and C849Y/WT rVWFs had a nearly normal multimeric pattern, whereas in hybrid R202W‐R1583Q/C849Y rVWF there was a loss of large/intermediate multimers. Conclusions: The propositus phenotype seems to be due to mutations R202W and C849Y, both affecting the VWF multimerization process and, for C849Y rVWF, intracellular survival. The absent triplet multimeric structure in the propositus and its reduction in his daughter appears to be related to the lack of ULVWF multimers, which mainly contribute to the formation of satellite bands.