Ribosome readthrough accounts for secreted full-length factor IX in hemophilia B patients with nonsense mutations

Authors

  • Mirko Pinotti,

    Corresponding author
    1. Dipartimento di Biochimica e Biologia Molecolare and LTTA Centre, Università di Ferrara, Italy
    • Department of Biochemistry and Molecular Biology, University of Ferrara, via Fossato di Mortara n 74, 44121Ferrara, Italy.
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    • First two authors have contributed equally to the work.

  • Pierpaolo Caruso,

    1. Dipartimento di Biochimica e Biologia Molecolare and LTTA Centre, Università di Ferrara, Italy
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    • First two authors have contributed equally to the work.

  • Alessandro Canella,

    1. Dipartimento di Biochimica e Biologia Molecolare and LTTA Centre, Università di Ferrara, Italy
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  • Matteo Campioni,

    1. Dipartimento di Biochimica e Biologia Molecolare and LTTA Centre, Università di Ferrara, Italy
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  • Giuseppe Tagariello,

    1. Servizio Trasfusionale, Centro Regionale per le Malattie del Sangue ed Emofilia, Ospedale di Castelfranco Veneto e Montebelluna, Castelfranco Veneto, Italy
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  • Giancarlo Castaman,

    1. Dipartimento di Terapie Cellulari ed Ematologia, San Bortolo Hospital, Vicenza, Italy
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  • Sofia Giacomelli,

    1. Dipartimento di Terapie Cellulari ed Ematologia, San Bortolo Hospital, Vicenza, Italy
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  • Donata Belvini,

    1. Servizio Trasfusionale, Centro Regionale per le Malattie del Sangue ed Emofilia, Ospedale di Castelfranco Veneto e Montebelluna, Castelfranco Veneto, Italy
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  • Francesco Bernardi

    1. Dipartimento di Biochimica e Biologia Molecolare and LTTA Centre, Università di Ferrara, Italy
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  • Communicated by John McVey

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. Hum Mutat 33:1373–1376, 2012. © 2012 Wiley Periodicals, Inc.

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