title: Characterization of an apparently synonymous F5 mutation causing aberrant splicing and factor V deficiency.
abstract: Coagulation factor V (FV) deficiency is a rare autosomal recessive bleeding disorder. We investigated a patient with severe FV deficiency (FV:C < 3%) and moderate bleeding symptoms. Thrombin generation experiments showed residual FV expression in the patient's plasma, which was quantified as 0.7 ± 0.3% by a sensitive prothrombinase-based assay. F5 gene sequencing identified a novel missense mutation in exon 4 (c.578G>C, p.Cys193Ser), predicting the abolition of a conserved disulphide bridge, and an apparently synonymous variant in exon 8 (c.1281C>G). The observation that half of the patient's F5 mRNA lacked the last 18 nucleotides of exon 8 prompted us to re-evaluate the c.1281C>G variant for its possible effects on splicing. Bioinformatics sequence analysis predicted that this transversion would activate a cryptic donor splice site and abolish an exonic splicing enhancer. Characterization in a F5 minigene model confirmed that the c.1281C>G variant was responsible for the patient's splicing defect, which could be partially corrected by a mutation-specific morpholino antisense oligonucleotide. The aberrantly spliced F5 mRNA, whose stability was similar to that of the normal mRNA, encoded a putative FV mutant lacking amino acids 427-432. Expression in COS-1 cells indicated that the mutant protein is poorly secreted and not functional. In conclusion, the c.1281C>G mutation, which was predicted to be translationally silent and hence neutral, causes FV deficiency by impairing pre-mRNA splicing. This finding underscores the importance of cDNA analysis for the correct assessment of exonic mutations.
authors.affiliation: Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands.
authors.forename: F
authors.initials: F
authors.lastname: Nuzzo
authors.affiliation: Department of Cardiologic, Thoracic and Vascular Sciences, 2nd Chair of Internal Medicine, University of Padua Medical School, Padua, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Bulato
authors.affiliation: Harold R. Roberts Hemophilia and Thrombosis Center, Chapel Hill, NC, USA.
authors.forename: B I
authors.initials: BI
authors.lastname: Nielsen
authors.affiliation: Harold R. Roberts Hemophilia and Thrombosis Center, Chapel Hill, NC, USA.
authors.forename: K
authors.initials: K
authors.lastname: Lee
authors.affiliation: Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands.
authors.forename: S J
authors.initials: SJ
authors.lastname: Wielders
authors.affiliation: Department of Cardiologic, Thoracic and Vascular Sciences, 2nd Chair of Internal Medicine, University of Padua Medical School, Padua, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Simioni
authors.affiliation: Harold R. Roberts Hemophilia and Thrombosis Center, Chapel Hill, NC, USA.|Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
authors.forename: N S
authors.initials: NS
authors.lastname: Key
authors.affiliation: Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands.
authors.forename: E
authors.initials: E
authors.lastname: Castoldi
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D002460:Cell Line; D005091:Exons; D005165:Factor V / Q000235:genetics*; D005166:Factor V Deficiency / Q000097:blood / Q000175:diagnosis / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D008297:Male; D009154:Mutation; D013917:Thrombin / Q000096:biosynthesis; D055815:Young Adult
chemical_list: D005165:Factor V; D013917:Thrombin
keywords: factor V deficiency; mRNA analysis; morpholino antisense oligonucleotide; splicing; synonymous mutation