Molecular characterization, recombinant protein expression, and mRNA analysis of type 3 von Willebrand disease: Studies of an Italian cohort of 10 patients

Abstract

Type 3 von Willebrand disease (VWD3) is characterized by unmeasurable von Willebrand factor (VWF) levels in plasma and platelets and severe but variable hemorrhagic symptoms. To identify and characterize the causal mutations, we screened 10 Italian patients with VWD3 by several techniques including Multiplex Ligation‐dependent Probe Amplification to identify large insertions and deletions, High Resolution Melting and PCR coupled with Sanger sequencing. Fourteen different mutations scattered throughout the VWF gene were identified, 10 of which were novel. As expected, most of these mutations caused null alleles: five were deletions (del exons 1–3, del exon 17, c.2157delA, c.2269delCT, and c.3940delG), three nonsense (p.Q1526X, p.E1549X, and p.C2448X) and three potential splice‐site mutations (c.658‐2A>G, c.7729+7C>T, and c.8155+1G>T). Three candidate missense mutations (p.C2184S, p.C2212R, and p.C2325S) were also identified. Missense mutations and the putative splice‐site defects were confirmed to be disease related by in vitro expression studies and mRNA analysis. None of these patients have developed alloantibodies against VWF. This study extends our previous finding that most of the mutations that we identified in VWD3 patients arise independently and are scattered throughout the entire VWF gene. Am. J. Hematol. 2012. © 2012 Wiley Periodicals, Inc.

Genetic Heterogeneity in a Large Cohort of Indian Type 3 von Willebrand Disease Patients

“…This is comparable to several other reports of mutation characterization in VWD patients, wherein different sensitivities (28–90%) have been reported using different mutation screening and direct DNA sequencing techniques [11], [31]–[33]. In two patients, second heterozygous mutation could not be identified, despite sequencing all the exons of VWF and MLPA analysis.…”

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)

“…Genomic DNA was extracted from peripheral blood with standard methods. PCR was performed to amplify the patients’ whole VWF coding region, including the corresponding intron–exon boundaries, and sequence analysis was carried out as previously reported . All oligonucleotides and PCR conditions of the following methods are available on request.…”

“…In previous studies, other splice-site mutations have been reported to cause exon skipping in VWD patients [8,9,15,20,[23][24][25]. Among these, mutation c.3538+1G>A, identified in a VWD2AIIE patient, was found to lead to skipping of exon 26 in vitro [8].…”

Cryptic non-canonical splice site activation is part of the mechanism that abolishes multimer organization in the c.2269_2270del von Willebrand factor

“…In vitro expression experiments confirmed that the c.2269_2270del mutation, hitherto considered a type 3 VWF defect(29,41), is actually associated with a residual synthesis of VWF due to the presence of RNAII and RNAIII forms. While RNAI (the prevalent VWF mRNA form) produced no rVWF, both RNAII and RNAIII produced a rVWF molecule characterized by the persistence of VWFpp, as shown by the slower migration of each oligomer, and the weaker FVIII binding capacity.…”