Only a few F9 nonsense mutations are responsive to drug-induced readthrough due to specific translation and protein structural constraints.
Re-insertion of the wild-type residue and gain-of-function effects account for functionally relevant readthrough.
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 readthrough-inducing drug geneticin of eleven 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 two (p.W240X, p.R384X) responded with a remarkable rescue of FIX activity. While the amounts of rescued full-length FIX protein for the p.W240X (~9% of rFIX-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: i) prevalent re-insertion of the authentic residue (tryptophan), reverting the nonsense effects, for the p.W240X, and ii) gain-of-function for the p.R384X, supported by the 4-fold increased activity of the most probable readthrough-mediated missense variant (rFIX-R384W). For most PTCs, impaired secretion/function produced by readthrough-mediated amino acid substitutions prevented a significant functional rescue, which requires combinations of favorable FIX mRNA sequence and protein features. This rational approach, applicable to other coagulation disorders, helps interpreting the poor response reported in the few investigated HB patients, and identifies candidate patients eligible for treatment.
- Submitted September 16, 2016.
- Accepted February 11, 2017.
- Copyright © 2017 American Society of Hematology
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