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

• Published in: Haematologica (Roma) 2008-10, Vol.93 (10), p.1505-1513
• Main Authors: Dall'Osso, Claudia, Guella, Ilaria, Duga, Stefano, Locatelli, Nadia, Paraboschi, Elvezia Maria, Spreafico, Marta, Afrasiabi, Abdolreza, Pechlaner, Christoph, Peyvandi, Flora, Tenchini, Maria Luisa, Asselta, Rosanna
• Format: Article
• Language: English
• Subjects: Adult; Animals; Base Sequence; Biological and medical sciences; Cell Line; Cercopithecus aethiops; Factor V - chemistry; Factor V - genetics; Factor V - metabolism; Factor V Deficiency - genetics; Factor V Deficiency - metabolism; Female; Hematologic and hematopoietic diseases; Humans; Male; Medical sciences; Middle Aged; Models, Molecular; Mutation - genetics; Platelet diseases and coagulopathies; Protein Structure, Tertiary; RNA Splicing - genetics; RNA, Messenger - genetics
• ISSN: 0390-6078; 1592-8721
• DOI: 10.3324/haematol.12934

1 Department of Biology and Genetics for Medical Sciences, University of Milan, Italy 2 A. Bianchi Bonomi, Hemophilia and Thrombosis Center, University of Milan and Department of Medicine and Medical Specialties, IRCCS Maggiore Hospital, Mangiagalli and Regina Elena Foundation, Milan, Italy 3 Hemostasis and Thrombosis Unit, Haematology Research Center, Shiraz Medical University of Science, Shiraz, Iran 4 Innsbruck Medical University, University Hospital, Department of Internal Medicine, Innsbruck, Austria Correspondence: Rosanna Asselta, Ph.D., Department of Biology and Genetics for Medical Sciences, University of Milan, via Viotti 3/5, 20133 Milan, Italy. E-mail: rosanna.asselta{at}unimi.it 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 - 8-mRNA). Interestingly, F5 - 8-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 - 8-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 th