INTRODUCTION The complete deficiency of factor VII (FVII), the protease triggering coagulation, is considered to be lethal. However, methodological limitations do not enable to investigate extremely low FVII expression levels. We investigated the FVII IVS6+1g/t change that, by altering the invariable +1 position of a donor splice site (5’ss), potentially represents a “”null mutation”. The IVS6+1g/t is frequent in Thailand FVII deficient patients and associated to life-threatening bleeding. METHODS FVII mRNA splicing and protein levels were investigated by expression in Hep3B and BHK cells of FVII minigenes, modified U1snRNAs and of FVII cDNA variants. RESULTS The IVS6+1g/t mutation mainly induced exon 6 skipping and, to a much lower extent, total and partial IVS6 retention, thus producing frame-shifts incompatible with correct FVII biosynthesis. We did not detect any trace of normal transcripts that would support a minimal FVII function in patients. Targeting the mutated 5’ss at the natural junction by modified U1-IVS6+1t did not rescue correct splicing to any extent. Intriguingly, capillary denaturing electrophoresis also identified traces of an in-frame 30bp deleted FVII mRNA resulting from the usage of an exonic cryptic 5’ss. Recognition of this cryptic site was improved (14% of the exon skipped transcript) by engineering U1snRNA. Upon in-vitro expression, the deleted FVII variant lacking 10 residues (Val158-Gln167) of the activation domain displayed, in fluorogenic functional assays in plasma systems, a low but detectable activity (0.05% of wt). CONCLUSION Re-programming mRNA processing by modified U1snRNAs able to induce cryptic 5’ss activation could represent a novel correction approach for null 5’ss mutations. Remarkably, compensatory aberrant splicing might explain residual FVII expression associated to a splicing mutation type that otherwise would result in a “null genetic condition”.
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|Titolo:||“Compensatory” aberrant splicing supports residual expression levels in severe coagulation factor VII deficiency|
|Autori interni:||BALESTRA, Dario|
|Data di pubblicazione:||2011|
|Rivista:||JOURNAL OF THROMBOSIS AND HAEMOSTASIS|
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