Readthrough-mediated functional suppression of homozygous nonsense mutations accounts for variable bleeding phenotypes in factor VII deficiency

BACKGROUND. Nonsense mutations cause premature stop codons that lead to the potential absence of the gene product. Whereas the rare homozygous nonsense mutations causing factor (F)VII deficiency may predict null conditions almost completely incompatible with life, they are associated with appreciable differences in hemorrhagic symptoms, possibly due to residual levels of full-length FVII arising from spontaneous readthrough. This process, based on misrecognition of premature stop codons, may induce the synthesis of variable levels of functional full-length proteins. The sequence context (namely, stop codon and downstream nucleotide) predicts a variable degree of readthrough efficiency in terms of amount of full-length protein produced. The protein outcome is also strictly dependent on the most probable substituted residues, ranging from the wild-type amino acid reinsertion to missense changes impairing secretion and function. The aim of the study was to experimentally evaluate the basal and drug-induced levels of FVII resulting from the homozygous p.Cys132X and p.Ser112X nonsense mutations that are associated with moderate or life-threatening symptoms, respectively, and are predicted to undergo readthrough with (132X) or without (112X) production of wild-type FVII. METHODS. We transiently expressed recombinant FVII (rFVII) nonsense and missense variants in human embryonic kidney 293 cells. Secreted FVII protein levels were evaluated by ELISA while functional levels were assessed through activated FX generation and coagulation assays. RESULTS. The levels of functional FVII produced by p.Cys132X and p.Ser112X mutants (rFVII-132X, 1.1%±0.2% of wild-type rFVII; rFVII-112X, 0.5%±0.1% of wild-type rFVII) were compatible with the occurrence of spontaneous readthrough. Treatment with G418, an aminoglycoside known to increase misrecognition of stop codons, allowed to magnify the occurrence of drug-induced readthrough, up to 12% of the wild-type value for the rFVII-132X nonsense variant. Previous studies indicated that TGA is suppressed by the incorporation of Trp>Cys>Arg, in order of frequency. To dissect the mechanism underlying the production of a functional full-length protein we expressed all the predicted missense variants arising from readthrough, which showed abolished (rFVII-132Trp/Arg) or reduced (rFVII-112Trp/Cys/Arg, 22–45% of wild-type levels) secretion and function. These data suggest that the appreciable rescue of p.Cys132X function was driven by reinsertion of the wild-type residue, whereas the minimal p.Ser112X function was explained by missense changes permitting FVII secretion and function. CONCLUSIONS. Our experimental findings support the notion that the extent of functional readthrough contributes to the variable bleeding phenotype in patients homozygous for F7 nonsense mutations, possibly preventing null conditions even for the most readthrough-unfavorable mutations.