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: F Nuzzo, C Bulato, B I Nielsen, K Lee, S J Wielders, P Simioni, N S Key, E Castoldi
• Journal: Haemophilia : the official journal of the World Federation of Hemophilia (Haemophilia) Vol. 21 Issue 2 Pg. 241-248 (Mar 2015) ISSN: 1365-2516 [Electronic] England
• PMID: 25470420 (Publication Type: Case Reports, Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2014 John Wiley & Sons Ltd.

Chemical References

• Factor V
• Thrombin

Topics

• Alternative Splicing
• Animals
• Cell Line
• Exons
• Factor V (genetics)
• Factor V Deficiency (blood, diagnosis, genetics)
• Gene Expression
• Humans
• Male
• Mutation
• Thrombin (biosynthesis)
• Young Adult