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Highlights: [•] Disease-causing missense mutations mainly impair protein biosynthesis and/or function. [•] The p.Y450C mutation in factor IX (FIX) provided a model to study their interplay. [•] The mutation in the carboxyl-terminus impairs both FIX protein secretion and activity. [•] The phenyl group at this relatively conserved position (c234) has a key role. [•] The differential effects have pathophysiological and evolutionary implications. [ABSTRACT FROM AUTHOR]

Drug-induced readthrough over premature stop codons (PTCs) is a potentially attractive therapy for genetic disorders, but a wide outcome variability has been observed. Through expression studies, we investigated the responsiveness to the read through-inducing drug geneticin of 11 rationally selected factor IX (FIX) nonsense mutations, present in 70% (324/469) of hemophilia B (HB) patients with PTCs. Among the predicted readthrough-permissive TGA variants, only 2 (p.W240X and p.R384X) responded with a remarkable rescue of FIX activity. The amounts of rescued full-length FIX protein for the p.W240X (~9% of recombinant FIX [rFIX]--wild-type [WT]) slightly exceeded activity (5.2 ± 0.6%). FIX antigen for the p.R384X (1.9 ± 0.3%) was remarkably lower than activity (7.5 ± 0.7%). Data indicate novel specific mechanisms producing functional rescue: (1) prevalent reinsertion of the authentic residue (tryptophan), reverting the nonsense effects for the p.W240X, and (2) gain-of-function for the p.R384X, supported by the fourfold increased activity of the most probable readthrough-mediated missense variant (rFIX-R384W). For most PTCs, impaired secretion/function produced by read through-mediated amino acid substitutions prevented a significant functional rescue, which requires combinations of favorable FIX messenger RNA (mRNA) sequence and protein features. This rational approach, applicable to other coagulation disorders, helps with interpreting the poor response reported in the few investigated HB patients, and identifies candidate patients eligible for treatment. [ABSTRACT FROM AUTHOR]