title: Ribosome readthrough accounts for secreted full-length factor IX in hemophilia B patients with nonsense mutations.
abstract: We investigated the spontaneous ribosome readthrough, virtually unexplored in genes encoding secreted proteins, over coagulation F9 nonsense mutations. Expression of recombinant factor IX (FIX) in eukaryotic cells demonstrated appreciable levels of secreted FIX molecules for the mutations p.R162* (5 ± 0.3% of rFIX-wt antigen levels), p.R294* (3.1 ± 1.1%) and p.R298* (2.5 ± 0.7%), but not for the p.L103*. Western blotting revealed a large proportion of truncated molecules, which correlated with small amounts of full-length FIX (rFIX-162*, ∼0.5%; rFIX-294*; and rFIX-298*, ∼0.2%). Western blotting of plasma from FIX deficient (Hemophilia B) patients revealed traces of full-length FIX for the p.R294* and p.R298* mutations, but not for the p.L103* mutation that triggered major FIX mRNA decay. The detection of full-length proteins has clinical implication, particularly for post-therapeutic immunological complications in Hemophilia. Data in patients' plasma and in vitro, obtained in the proper protein context, support a ribosome readthrough gradient, consistent with its predicted determinants of efficiency.
authors.affiliation: Dipartimento di Biochimica e Biologia Molecolare and LTTA Centre, Università di Ferrara, Italy. pnm@unife.it
authors.forename: Mirko
authors.initials: M
authors.lastname: Pinotti
authors.forename: Pierpaolo
authors.initials: P
authors.lastname: Caruso
authors.forename: Alessandro
authors.initials: A
authors.lastname: Canella
authors.forename: Matteo
authors.initials: M
authors.lastname: Campioni
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Tagariello
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Castaman
authors.forename: Sofia
authors.initials: S
authors.lastname: Giacomelli
authors.forename: Donata
authors.initials: D
authors.lastname: Belvini
authors.forename: Francesco
authors.initials: F
authors.lastname: Bernardi
mesh_terms: D015153:Blotting, Western; D018389:Codon, Nonsense / Q000235:genetics*; D005164:Factor IX / Q000235:genetics / Q000378:metabolism*; D006207:Half-Life; D002836:Hemophilia B / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011237:Predictive Value of Tests; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics* / Q000378:metabolism
chemical_list: D018389:Codon, Nonsense; D012333:RNA, Messenger; D011994:Recombinant Proteins; D005164:Factor IX