The F7 p.Val22Ile missense mutation affects splicing and can be counteracted by a compensatory U1snRNA

Background: Aberrant splicing is a frequent but still underestimated pathogenic mechanism. We studied the F7 c.64G→A mutation, associated with factor VII (FVII) deficiency (activity 6- 8%). It occurs at -1 position of the 5' splice site (5'ss) of exon 1a and predicts the V22I change in the FVII pre-peptide, which guides nascent FVII into the endoplasmic reticulum (ER). Aims: To elucidate the molecular mechanisms leading to FVII deficiency Methods: Expression of i) FVII cDNA alone or ii) fused with green fluorescent protein (FVIIGFP) and iii) of the splicing-competent FVII cDNA in Baby Hamster Kidney (BHK) cells. Evaluation of splicing patterns (RT-PCR), intracellular trafficking (fluorescent microscopy) and secreted FVII protein (Western blot, ELISA) and activity (FXa generation assays). Results: Expression of the FVII variant resulted in secreted FVII protein/activity levels comparable with FVII . Consistently, the FVII -GFP variant showed an unaltered intracellular trafficking to ER. To evaluate splicing all introns were, partially or integrally, included into the FVII cDNA (splicingcompetent, SC-F7 ). SC-F7 expression in BHK cells showed a F7 splicing pattern that resembled that in human hepatoma cells (HepG2), with the presence of the alternative exon 1b in ~11% of transcripts. Consistently, functional FVII was clearly detectable in medium. The 64G→A change (SC-F7 ) remarkably reduced the proportion of correctly spliced transcripts (8.0±1.3%) as compared with the SC-F7 (43.7±3.5%), and levels of secreted protein (2.1±2.1%). Moreover, a spliceosomal U1snRNA variant with improved complementarity for the mutated 5'ss rescued splicing (up to 33.7±7.2% of correct transcripts) and, consistently, remarkable increased secreted FVII protein (57.0±20.6%) and activity (69.8±22.7%) levels. Conclusions: Data demonstrate that the c.64G→A mutation affects F7 pre-mRNA splicing by impairing 5'ss-U1snRNA interaction. The entire splicing-competent FVII construct offers a novel tool to evaluate putative F7 splicing mutations, both for diagnostic and therapeutic (RNA-based) purposes.