von Willebrand disease type 1 mutation p.Arg1379Cys and the variant p.Ala1377Val synergistically determine a 2M phenotype in four Italian patients.

INTRODUCTION: We characterized five patients affected with von Willebrand disease (VWD) carrying the p.Arg1379Cys mutation. One was diagnosed as VWD type 1 and four as type 2M. The 2M patients also have the variant p.Ala1377Val in cis with p.Arg1379Cys. AIM: To evaluate the role of p.Ala1377Val and p.Arg1379Cys von Willebrand factor (VWF) variants to explain patients' phenotype. METHODS: Conventional phenotype tests were used to evaluate patients' plasma and platelets. Direct sequence analysis of exon 28 was carried out. The allele frequency of p.Ala1377Val was evaluated using online database. pcDNA3.1-VWF-WT and mutant (A1377V, R1379C and A1377V-R1379C) expression vectors were transiently transfected in HEK293 cells. The capacity of WT and mutant recombinant (r)VWF (along with patients' plasma VWF) to bind glycoprotein Ibα (GpIbα) were evaluated, using two ELISA assays. One with a wild-type (WT) recombinant (r)GpIbα at increasing ristocetin concentrations (from 0 to 1.50 mg mL-1 ) and the other with a gain-of-function mutant rGpIbα (VWF:GPIbM). RESULTS: The substitution c.4130C>T (p.Ala1377Val) was reported as rare variant in online databases. At 0.25 mg mL-1 of ristocetin, WT, A1377V and R1379C showed 6, 7.5 and 12-fold increased binding to rGpIbα, respectively. A1377V-R1379C rVWF showed no increased binding to rGpIbα at the same ristocetin concentration and reached the highest binding, of only 3-fold increased, at 1.50 mg mL-1 of ristocetin. The VWF:GPIbM showed strongly reduced values for the A1377V-R1379C rVWF and the 2M patients' plasma. CONCLUSION: Our study showed that the presence of both p.Ala1377Val and p.Arg1379Cys mutations (synergistic effect) abolishes the binding of rVWF to rGpIbα, explaining patients' 2M phenotype.

Evaluation of an heterogeneous group of patients with von Willebrand disease using an assay alternative to ristocetin induced platelet agglutination.

BACKGROUND: Diagnosis of von Willebrand disease (VWD) type 2 usually relies on the discrepancy between the von Willebrand factor (VWF) ristocetin cofactor activity (VWF:RCo) and VWF antigen (VWF:Ag). Type 2B patients can be discriminated from other qualitative VWD variants by using ristocetin-induced platelet agglutination (RIPA) test. The major limitation of RIPA is the requirement of fresh blood sample. OBJECTIVES: In this study, we evaluated the VWF gain-of-function mutant GPIb binding (VWF:GPIbM) and VWF:RCo assays to investigate whether the VWF:GPIbM/VWF:RCo ratio was able to identify the type 2B variant among an heterogeneous VWD population, previously characterized following the ISTH-SSC guidelines. PATIENTS/METHODS: Seventy-six VWD patients and 31 healthy subjects were evaluated by using VWF:Ag, VWF:RCo, and VWF:GPIbM assays. RESULTS: The mean (minimum-maximum values) VWF:GPIbM/VWF:RCo ratio was higher in type 2B patients (2.53, 0.84-6.11) than in healthy controls (1.05, 0.87-1.34), type 1 (0.85, 0.51-1.15), 2A (1.20, 0.36-2.82), and 2M (1.07, 0.91-1.38) (P < 0.0001). Type 2B variants were divided into four groups (A, B, C, and D) according to their different multimeric patterns. The mean value of the VWF:GPIbM/VWF:RCo ratio in the four groups showed an increasing trend from group A (1.08) to D (3.69), proportional to the loss of high molecular weight multimers. Among 32 type 2B patients, previously diagnosed with RIPA, 8 (mainly with a type I New York/Malmö phenotype) were not confirmed using the VWF:GPIbM/VWF:RCo ratio. CONCLUSIONS: Whenever the RIPA test is not feasible, the VWF:GPIbM/VWF:RCo ratio might help to identify severe type 2B VWD patients.

A synonymous (c.3390C>T) or a splice-site (c.3380-2A>G) mutation causes exon 26 skipping in four patients with von Willebrand disease (2A/IIE).

INTRODUCTION: We characterized four unrelated patients with von Willebrand disease type 2A/IIE, sharing the same von Willebrand factor (VWF) in-frame deletion (p.[P1127_G1180delinsR];[=]) resulting from exon 26 skipping (Δ26). OBJECTIVES: To identify the VWF mutations and how they caused the mRNA splicing alteration, to evaluate the deletion by in vitro expression studies, and to assess whether or not the heterogeneity of the patients' phenotype might be related to a different degree of expression of the deleted subunit in patient plasma VWF. METHODS: Sequence analysis was performed with patient genomic DNA and platelet mRNA. Semiquantitative RT-PCR was also carried out to compare the expression of the wild-type (WT) and Δ26 alleles in the four patients. In silico analysis was performed with prediction splicing programs. Expression studies were performed to evaluate mutant recombinant VWF (rVWF) (Δ26 and Δ26/WT) as compared with WT rVWF. RESULTS: Three patients shared the synonymous single-nucleotide substitution (SSS) c.[3390C>T];[=], whereas the novel mutation c.[3380-2A>G];[=] was present in the fourth patient. Semiquantitative RT-PCR of platelet mRNA revealed a different ratio of the WT and Δ26 alleles in the patients, consistent with the different VWF:FVIIIB values present in patient plasma. Expression studies confirmed reduced VWF-FVIII binding of rVWF-Δ26/WT. CONCLUSIONS: SSS can induce alternative splicing, and those like c.3390C>T, which impact on the poorly conserved splicing regulatory elements, are difficult to predict, so that their role can be evaluated only by mRNA analysis. Moreover, these mutations seem to have different effects on the efficiency of alternative splicing, producing heterogeneous VWF variants among the four patients.