Expression studies of mutant factor VIII alleles with premature termination codons with regard to inhibitor formation

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Expression studies of mutant factor VIII alleles with premature termination codons with regard to inhibitor formationM. A. Zimmermann1,*, J. Oldenburg2, C. R. Müller1 andS. Rost1Article first published online: 7 MAR 2014

DOI: 10.1111/hae.12388

© 2014 John Wiley & Sons Ltd

Issue

HaemophiliaVolume 20, Issue 3, pages e215–e221, May 2014

Additional Information

How to CiteZimmermann, M. A., Oldenburg, J., Müller, C. R. and Rost, S. (2014), Expression studies of mutant factor VIII alleles with premature termination codons with regard to inhibitor formation. Haemophilia, 20: e215–e221. doi: 10.1111/hae.12388

Author Information1

Department of Human Genetics, University of Würzburg, Würzburg, Germany

2

Institute of Experimental Haematology and Transfusion Medicine, University of Bonn, Bonn, Germany

* Correspondence: Melanie A. Zimmermann, Department of Human Genetics, University of Würzburg, Biocenter, Am Hubland, 97074 Würzburg, Germany.
Tel: +49 931 3181278; fax: +49 931 3184069;
e-mail: melanie.zimmermann@uni-wuerzburg.de

Publication HistoryIssue published online: 15 APR 2014Article first published online: 7 MAR 2014Manuscript Accepted: 27 JAN 2014Funded byBaxter Deutschland GmbH SEARCH Search Scope All contentPublication titlesIn this journalIn this issue Search String Advanced >Saved Searches > SEARCH BY CITATION Volume: Issue: Page: ARTICLE TOOLSGet PDF (216K)Save to My ProfileE-mail Link to this ArticleExport Citation for this ArticleGet Citation AlertsRequest Permissions AbstractArticleReferencesCited By View Full Article (HTML) Enhanced Article (HTML) Get PDF (216K) Keywords:expression;factor VIII;Haemophilia;Inhibitor;nonsense mutations;premature terminationSummary

About 10% of mutations in haemophilia A cases generate a premature termination codon in the factor VIII gene (F8). Upon therapeutic FVIII substitution, it was noted that the risk of developing inhibitors is higher when the nonsense mutation is located in the light chain (LC) of the factor VIII (FVIII) protein than in the heavy chain (HC). We analysed the impact of six different nonsense mutations distributed over the six FVIII domains on recombinant FVIII expression to elucidate the process of inhibitor formation in haemophilic patients. Full-length F8 mRNA was transcribed from all constructs despite the presence of nonsense mutations. Polyclonal antigen assays revealed high antigen levels in transfection experiments with constructs truncated in LC whereas low antigen was detected from constructs truncated in HC. Those results were supported by FVIII localization experiments. These findings suggest that F8 transcription occurs in a usual way despite nonsense mutations, whereas translation appears to be interrupted by the premature stop codon. We hypothesize that the inclusion of the B domain enables proteins truncated in LC to accumulate in the ER. Proteins truncated in HC are mainly degraded or may pass through the ER and be secreted into the blood circulation, thus presumably preventing inhibitor formation after therapeutic FVIII substitution. The LC is known to have higher immunogenicity than the HC. Moreover, translation of the F8B gene comprising F8 exons 23–26 may be dependent on the position of the premature stop codon and thus contributes to the immune response of truncated FVIII proteins.

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