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J Thromb Haemost. 2011 Apr;9(4):679-88. doi: 10.1111/j.1538-7836.2011.04204.x.

The study of the effect of splicing mutations in von Willebrand factor using RNA isolated from patients' platelets and leukocytes.

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1
Unitat de Diagnòstic i Teràpia Molecular, Banc de Sang i Teixits, Barcelona, Spain.

Abstract

BACKGROUND:

In von Willebrand factor (VWF) the effect of mutations potentially affecting mRNA processing or splicing is less predictable than that of other mutations (e.g. nonsense or missense substitutions). Bioinformatic tools can provide a valuable means to determine the consequences of potential splice site mutations (PSSM), but functional studies are mandatory to elucidate the true effect of the variation detected.

OBJECTIVES, PATIENTS AND METHODS:

After identification of PSSM in VWD patients, we began a systematic study of their in vivo effect in RNA extracted from the patients' platelets and leukocytes.

RESULTS AND CONCLUSIONS:

Thirteen pairs of primers were designed for full amplification of VWF mRNA by RT-PCR that, after sequencing of aberrant products, enabled elucidation of the PSSM consequences for mRNA processing. This procedure was used to study seven different PSSM identified in four patients demonstrating diverse molecular mechanisms such as exon skipping (c.533-2A>G and c.8155+3G>C) and the activation of a cryptic splice site (c.7730-1G>C). No visible effect was evident for c.1533+15G>A and c.5170+10C>T and the consequence of c.[546G>A;7082-2A>G] was hidden by nonsense-mediated mRNA decay (NMD). Results were compared with in silico predictions of four splice-site analysis tools. We demonstrate selective degradation of VWF mRNA bearing PSSM by NMD for several mutations, which suggests that NMD represents a general mechanism for truncating mutations in VWF. Furthermore, because NMD efficiency varies between cell types, use of RNA from both platelets and leukocytes for in vivo study of VWF PSSM offers complementary results, particularly in cases in which NMD occurs in the allele carrying the mutation.

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