Prevalence of type 2b ‘Malmö/New York’ von Willebrand disease in Italy: the role of von Willebrand factor gene conversion

von Willebrand disease type 2B (VWD2B) is due to a unique gain-of-function variant of von Willebrand factor (VWF) that spontaneously interacts with circulating platelets, usually resulting in loss of VWF high molecular weight multimers (HMWM) in plasma and, in most cases, low platelet counts, especially after stress situations [1,2]. Diagnosis of VWD2B is based on heightened ristocetin-induced platelet aggregation (RIPA) in platelet-rich plasma (PRP). VWD2B Malmö [3] or New York [4] , previously reported as type I, is associated with increased RIPA at low concentrations of ristocetin but normal HMWMand no thrombocytopenia after stress situations. This peculiar VWD2B variant is caused by the mutation 3797C>T (P1266L) [5] in the VWF gene [6]. However, the majority of previously reported patients carrying the mutation P1266L show more than one nucleotide substitution [5,7–10], all matching the published VWF pseudogene sequence [11]. This finding, first described in VWD by Eikenboom et al. [7], has been explained by amechanism of gene conversion between the VWF gene and its pseudogene. The aims of this study were to determine the prevalence of this mutation in a large cohort of VWD2B patients enrolled in the Italian Registry of VWD and to evaluate whether or not gene conversions play a role in generating the mutations identified. Criteria for VWD2B were those recommended by the International Society on Thrombosis and Haemostasis Scientific and StandardizationCommittee VWFSubcommittee (ISTH-SSC on VWF) [2]. RIPA and multimeric analyses were performed as previously reported [12,13]. Patients bleeding severity score was obtained using a detailed questionnaire [14]. Platelet counts had been obtained over 2 years at baseline and after physiologic (pregnancy) or pathological (infections, surgeries) situations or by the use of desmopressin. Among 1234 patients enrolled in the Italian Registry, 66 (35 unrelated families) were diagnosed with VWD2B because of RIPA < 0.7 mg mL (normal range 0.7–1.2 mg mL). DNA sequence analysis was performed using oligonucleotides specifically designed to amplify selectively the VWF gene. The 5¢ portion of exon 28 encoding for the VWF A1 domain was sequenced in all VWD2B cases with a normal VWFmultimeric pattern. Four unrelated families (13 individuals) had a normal multimeric pattern in plasma and no thrombocytopenia before and after stress situations. Mutation 3923G>T (R1308L) was responsible for this phenotype in one family (five patients), and has been documented in a previous study [15]. In the remaining three families (seven patients) more than one mutation was found, but all patients shared a substitution of proline 1266. The following nucleotide changes were identified: 3686T>G (V1229G), 3692A>C (N1231T), 3735G>A, 3789G>A and 3797C>T (P1266L) in family A (three patients), 3789G>A and 3797C>T (P1266L) in family B (two patients) and 3692A>C (N1231T), 3789G>A and 3797C>A (P1266Q) in family C (two patients) (Fig. 1). Nucleotide numbers are reported according to the VWF cDNA sequence [16]. The P1266L/Q genotypes correlate with the patients bleeding history and laboratory tests. Patients bleeding severity score was high (mean 2.85, range 1–6), although less than in other VWD2B variants (8, range 4–14; normal range 0 to )1)[14]. These lower bleeding severity scores are consistent with the mildly reduced or normal VWF levels and activities, reported herewith as mean values (ranges): VWF: Ag 56.4 (30– 73) IU dL, and VWF:RCo 47 (21–66) IU dL, increased RIPA 0.53 (0.40–0.70) mg mL, presence of the HMWMand normal platelet count. We have further investigated additional causes for the bleeding tendency of these patients. However, only in family A did two patients show mildly reduced platelet secretion after stimuli with ADP, as reported by others [17]. VWF:CB (mean values 56.8, 48–64 IU dL) could be tested only in patients with mutation P1266L, and were similar to those of VWF:RCo (56.8, 45–66 IU dL), at variance with the discrepant values reported in VWD2B patients carrying the R1308L mutation [15]. Correspondence: Luciano Baronciani, A. Bianchi Bonomi Hemophilia and Thrombosis Center, Foundation IRCCS Maggiore Policlinico Hospital, Mangiagalli, Regina Elena Via Pace 9, 20122 Milan, Italy. Tel.: +39 2 55 03 53 41; fax: +39 2 55 03 53 47. E-mail: luciano.baronciani@unimi.it