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

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 the identification of three alternatively spliced F5 transcripts.

Key words: factor V deficiency, F5, splicing mutations, nonsense-mediated mRNA decay, exon skipping, premature termination codons.