Abstract
Background: Type 2B von Willebrand disease (VWD) is due to a unique gain-of-function variant of von Willebrand factor (VWF) that spontaneously interacts with circulating platelets resulting in loss of VWF high molecular weight multimers (HMWM) in plasma and reduced platelet counts, in most cases. Diagnosis of 2B is based on increased ristocetin-induced platelet aggregation (RIPA) in patient’s platelet rich plasma (PRP). Type 2B “Malmo or New York” is caused by the mutation P1266L that is associated with increased RIPA, but with normal multimeric distribution in plasma and no thrombocytopenia.
Aims: To determine the incidence and the molecular defects of this rare VWF variant among a large population of VWD patients regularly followed at two Northern Italian Hemophilia Centers.
Methods: Criteria for VWD type 2B were those recommended by the ISTH-SSC-SC on VWF. RIPA and multimeric analysis were performed according to standardized methods. Bleeding severity score (BSS) was calculated in all patients enrolled in this study. Sequence analysis of the portion of exon 28 encoding for the A1 domain was performed to identify nucleotide substitutions in all cases where RIPA was <0.8 mg/ml.
Results: 74 patients from 16 different families satisfied the criteria for VWD type 2B. Seven patients from 3 unrelated families clearly showed normal multimeric distribution in plasma and no thrombocytopenia also after stimuli. Clinical and laboratory parameters of the seven affected members are reported as mean values (ranges): BSS=2.5 (2–4); RIPA=0.56 (0.5–0.7) mg/ml; platelet count=255 x 10^9/L (172–353); VWF:RCo=46 U/dL (21–66); VWF:Ag=53 U/dL (30–67); FVIII=61 U/dL (54–76). Patients from family A showed five distinct substitutions (3686T>G, 3692A>C, 3735G>A, 3789G>A, 3797C>T), patient B1 two (3789G>A, 3797C>T) and patient from family C three (3692A>C, 3789G>A, 3797C>A). Two of these were silent, whereas the other caused the following amino acid changes: three in family A (V1229G, N1231T, P1266L), one in B1 (P1266L) and two in family C (N1231T, P1266Q). Mutations V1229G and N1231T have already been reported, whereas mutation P1266Q is novel. The substitutions found in families A and B clearly correspond to the pseudogene sequence, suggesting gene conversion into VWF gene. In family C, the nucleotide mutation causing P1266Q substitution is not consistent with this mechanism: moreover the patients do not show the substitution 3735G>A found in the pseudogene.
Conclusion: We confirm that the substitution of proline 1266 with leucine appears to be the main cause of type 2B “Malmo/New York” phenotype while its replacements with a glutamine apparently results in lower VWF activities in family C. Based on these data, gene conversion appears to be more frequent than expected and capable to generate different set of mutations.
Cosegregation of von Willebrand factor gene polymorphisms and possible germinal mosaicism in type IIB von Willebrand disease
