title: Specific factor IX mRNA and protein features favor drug-induced readthrough over recurrent nonsense mutations.
abstract: 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 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 readthrough-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.
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy.
authors.forename: Alessio
authors.identifier: 0000-0002-6113-2694
authors.initials: A
authors.lastname: Branchini
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy.
authors.forename: Mattia
authors.initials: M
authors.lastname: Ferrarese
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy.
authors.forename: Matteo
authors.initials: M
authors.lastname: Campioni
authors.affiliation: Center for Bleeding Disorders and Coagulation, Department of Oncology, Careggi University Hospital, Florence, Italy; and.
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Castaman
authors.affiliation: Centre for Haemostasis and Thrombosis, Haematology Section, Department of Medical Sciences, University of Ferrara, Ferrara, Italy.
authors.forename: Rosella
authors.initials: R
authors.lastname: Mari
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Bernardi
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy.
authors.forename: Mirko
authors.initials: M
authors.lastname: Pinotti
mesh_terms: D019943:Amino Acid Substitution; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D018389:Codon, Nonsense; D005164:Factor IX / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D005839:Gentamicins / Q000494:pharmacology*; D057809:HEK293 Cells; D002836:Hemophilia B / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D006801:Humans; D014176:Protein Biosynthesis / Q000187:drug effects*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism
chemical_list: D018389:Codon, Nonsense; D005839:Gentamicins; D011994:Recombinant Proteins; D014364:Tryptophan; D005164:Factor IX; D001120:Arginine; C010680:antibiotic G 418