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.

Synonymous Mutations Add a Layer of Complexity in the Diagnosis of Human Osteopetrosis

“…Similar findings have been reported in other disorders, (26)(27)(28)(29)(30)(31)(32) also including skeletal defects (33)(34)(35) ; nonetheless, the contribution of synonymous mutations to human pathologies is likely highly underestimated at present. Although performing functional evaluations of all the synonymous changes in an individual exome is not cost-effective, it might be worth assessing the effect of those located in genes already known to be associated with diseases.…”

Diagnostic high-throughput sequencing of 2,390 patients with bleeding, thrombotic and platelet disorders

“…With the use of HTS, deep intronic variants have been identified and apparent silent variants in BPD genes have been shown to alter splicing. [30][31][32][33] Variants disrupting transcription regulatory motifs located in gene promoters and enhancer regions have also been associated with BPDs and with the recent mapping of endothelial and blood cell specific enhancers, it is likely that more variants located in these regions will be identified as associated with disease in the near future. [34][35][36] Nevertheless, due to the challenges in interpretation of non-coding variants we have only targeted and prioritised intronic and regulatory variants if previously associated with disease.…”

Molecular and functional characterization of the BMPR2 gene in Pulmonary Arterial Hypertension

“…Additional functional analyses are necessary to establish the mechanism underlying disease since not all the mutations associated with splicing alterations are classified as such after in silico analysis and vice versa. In this sense, minigene assay has been reported as a good approach to evaluate possible splicing alterations related to all these variants 19, 27, 28, 42, 44, 45 .…”