Abstract Number: PB1188
Meeting: ISTH 2020 Congress
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 (FVII-GFP) 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 FVII22I variant resulted in secreted FVII protein/activity levels comparable with FVIIwt. Consistently, the FVII22I-GFP variant showed an unaltered intracellular trafficking to ER.
To evaluate splicing all introns were, partially or integrally, included into the FVII cDNA (splicing-competent, SC-F7wt). SC-F7wt 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-F764A) remarkably reduced the proportion of correctly spliced transcripts (8.0±1.3%) as compared with the SC-F7wt (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.
To cite this abstract in AMA style:Ferraresi P, Rizzotto L, Branchini A, Baroni M, Balestra D, Bernardi F, Pagani F, Pinotti M. The F7 p.Val22Ile Missense Mutation Affects Splicing and Can Be Counteracted by a Compensatory U1snRNA [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/the-f7-p-val22ile-missense-mutation-affects-splicing-and-can-be-counteracted-by-a-compensatory-u1snrna/. Accessed September 29, 2023.
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