Characterization of an apparently synonymous F5 mutation causing aberrant splicing and factor V deficiency

F. Nuzzo, C. Bulato, B. I. Nielsen, K. Lee, S. J. Wielders, P. Simioni, N. S. Key, E. Castoldi*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review


Coagulation factor V (FV) deficiency is a rare autosomal recessive bleeding disorder. We investigated a patient with severe FV deficiency (FV:CC, 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.
Original languageEnglish
Pages (from-to)241-248
Issue number2
Publication statusPublished - Mar 2015


  • factor V deficiency
  • morpholino antisense oligonucleotide
  • mRNA analysis
  • splicing
  • synonymous mutation

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