A 3' untranslated region (3' UTR) variation on the factor VII (FVII) gene changes completely the phenotype due to the Arg277Cys mutation in a patient with Type I FVII deficiency

Abstract number: OC271

Peyvandi^* F., Garagiola^* I., Palla^* R., Marziliano† N., Bader^* R., Bajetta^* M. T., Mannucci^* P. M.

†Applera Italia, Molecular Biology Laboratory, Italy ^*University of Milan, Italy;

Mutations affecting interface residues of the protease domain indicate that TF allosterically influences the active site of FVIIa, stabilizing the labile zymogen to active enzyme transition. We report the molecular characterization of a patient with severe Type I FVII deficiency (FVII:C < 1% and FVII:Ag 1%) carrying the Arg277Cys mutation and the Arg353Gln polymorphism in homozygous state. The substitution of Arg277 by Cys located in the protease domain was studied by alanine scanning mutagenesis which showed a combined impairment of both TF binding and proteolytic activity of FVIIa. In vitro expression analysis of FVIICys277 confirmed the alanine scanning mutagenesis results. Moreover a pulse-chase labeling study demonstrated an impaired secretion pathway with an intracellular accumulation due to the Arg277Cys mutation in association with the Arg353Gln polymorphism. In fact this experiment reported in cell supernatant FVII:C-value of 5% and FVII:Ag of 30%. These results surprisingly did not confirm the patient's phenotype. In order to explain this discrepancy, we carried out a detail sequencing analysis of the FVII gene which showed 5 additional gene variations (73G/A, -401G/T, -122C/T, -323 + 10 bp) all present in the homozygous state. This analysis revealed also a novel 2 bp (2a) insertion at nucleotide 11293 located on the 3' untranslated region (UTR), the first mutation in this region of the FVII gene. This mutation has been analyzed in a population-based study of 113 Italian healthy subjects and was present in 15% of subjects. In vitro expression studies showed a 43% reduction of FVII:C and a 37% reduction of FVII:Ag in cells transfected by 2a insertion construct. Gene expression studies of FVII wild-type (FVIIWT) and FVII + 2a insertion constructs were done by the RT-PCR real time which demonstrated a 40% reduced level of FVII + 2a transcription. Finally we performed an in vitro expression study using a construct containing the novel 2a insertion, the Arg277Cys mutation and also the Arg353Gln polymorphism. The results showed that the levels of FVII:C and FVII:Ag were < 1% and 1%, respectively, confirming the patient's plasma data. In conclusion we characterized a patient with FVII deficiency carrying multiple mutations and a novel 2a insertion mutation localized at 3'UTR leading to a reduction of FVII gene expression by decreased transcription efficiency causing type I FVII deficiency.