Molecular characterization of three novel splicing mutations causing factor V deficiency and analysis of the F5 gene splicing pattern

Claudia Dall'Osso, Ilaria Guella, Stefano Duga, Nadia Locatelli, Elvezia Maria Paraboschi, Marta Spreafico, Abdolreza Afrasiabi, Christoph Pechlaner, Flora Peyvandi, Maria Luisa Tenchini, Rosanna Asselta

Research output: Contribution to journalArticlepeer-review


Background: Factor V deficiency is a rare autosomal recessive hemorrhagic disorder, associated with bleeding manifestations of variable severity. In the present study, we investigated the molecular basis of factor V deficiency in three patients, and performed a comprehensive analysis of the factor V gene (F5) splicing pattern. Design and Methods: Mutational screening was performed by DNA sequencing. Wild-type and mutant F5 mRNA were expressed by transient transfection in COS-1 cells, followed by reverse-transcriptase polymerase chain reaction and sequencing. Real-time reverse-transcriptase polymerase chain reaction was used to evaluate degradation of mRNA carrying premature termination codons. Results: Mutational screening identified three hitherto unknown splicing mutations (IVS8+6T>C, IVS21+1G>A, and IVS24+1_+4delGTAG). Production of mutant transcripts in COS-1 cells demonstrated that both IVS21+1G>A and IVS24+1_+4delGTAG cause the activation of cryptic donor splice sites, whereas IVS8+6T>C causes exon-8 skipping (F5-D8-mRNA). Interestingly, F5-D8-mRNA was also detected in wild-type transfected samples, human liver, platelets, and HepG2 cells, demonstrating that F5 exon-8 skipping takes place physiologically. Since F5-D8-mRNA bears a premature termination codons, we investigated whether this transcript is subjected to nonsense-mediated mRNA decay degradation. The results confirmed the involvement of nonsense-mediated mRNA decay in the degradation of F5 PTC+ mRNA. Moreover, a comprehensive analysis of the F5 splicing pattern led to the identification of two in-frame splicing variants resulting from skipping of exons 3 and 5-6. Conclusions: The functional consequences of three splicing mutations leading to FV deficiency were elucidated. Furthermore, we report the identification of three alternatively spliced F5 transcripts.

Original languageEnglish
Pages (from-to)1505-1513
Number of pages9
Issue number10
Publication statusPublished - Oct 2008


  • Exon skipping
  • F5
  • Factor V deficiency
  • Nonsense-mediated mRNA decay
  • Premature termination codons
  • Splicing mutations

ASJC Scopus subject areas

  • Hematology


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