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Molecular characterization, recombinant protein expression, and mRNA analysis of type 3 von Willebrand disease: Studies of an Italian cohort of 10 patients
Author(s) -
Solimando Maria,
Baronciani Luciano,
La Marca Silvia,
Cozzi Giovanna,
Asselta Rosanna,
Canciani Maria Teresa,
Federici Augusto B.,
Peyvandi Flora
Publication year - 2012
Publication title -
american journal of hematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.456
H-Index - 105
eISSN - 1096-8652
pISSN - 0361-8609
DOI - 10.1002/ajh.23265
Subject(s) - missense mutation , von willebrand disease , sanger sequencing , microbiology and biotechnology , exon , von willebrand factor , multiplex ligation dependent probe amplification , nonsense mutation , biology , genetics , mutation , gene , high resolution melt , genotype , immunology , platelet
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.