Clustered F8 missense mutations cause hemophilia A by combined alteration of splicing and protein biosynthesis/activity

Donadon, Irving, McVey, John, Garagiola, Isabella, Branchini, Alessio, Mortarino, Mimosa, Peyvandi, Flora, Bernardi, Francesco and Pinotti, Mirko (2017) Clustered F8 missense mutations cause hemophilia A by combined alteration of splicing and protein biosynthesis/activity Haematologica, 103 (2). pp. 344-350.

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Abstract

Dissection of pleiotropic effects of missense mutations, rarely investigated in inherited diseases, is fundamental to understanding genotype-phenotype relationships. Missense mutations might impair mRNA processing in addition to protein properties. As a model for hemophilia A we investigated the highly prevalent F8 c.6046c>t/p.R2016W (exon 19) mutation. In expression studies exploiting lentiviral vectors, we demonstrated that the amino acid change impairs both factor VIII (FVIII) secretion (antigen 11.0±0.4% of wild-type) and activity (6.0±2.9%). Investigations in patients’ ectopic F8 mRNA and with minigenes showed that the corresponding nucleotide change also decreases correct splicing to 70±5%, which is predicted to lower further FVIII activity (4.2±2%), consistently with patients’ levels (<1-5%). Masking the mutated exon 19 region by antisense U7snRNA supported the presence of a splicing regulatory element, potentially affected by several hemophilia A-causing missense mutations. Among these, the c.6037g>a (p.G2013R) reduced exon inclusion to 41±3% and the c.6053a>g (p.E2018G) to 28±2%, similarly to a variant affecting the 5’ splice site (c.6113a>g, p.N2038S, 26±2%), which displayed normal protein features upon recombinant expression. The p.G2013R remarkably reduced both antigen (7.0±0.9%) and activity (8.4±0.8%), while the p.E2018G produced a dysfunctional molecule (antigen, 69.0±18.1%; activity, 19.4±2.3%). In conclusion, differentially altered mRNA and protein patterns produce a gradient of residual activity, and clarify genotype-phenotype relationships. Data detail pathogenic mechanisms that, only in combination, account for moderate/severe disease forms, which in turn determine the mutation profile. Altogether we provide a clear example of interplay between mRNA and protein mechanisms of disease that certainly operate in shaping many other inherited disorders.

Item Type:	Article
Divisions :	Faculty of Health and Medical Sciences > School of Biosciences and Medicine
Authors :	Name Email ORCID Donadon, Irving McVey, John j.mcvey@surrey.ac.uk Garagiola, Isabella Branchini, Alessio Mortarino, Mimosa Peyvandi, Flora Bernardi, Francesco Pinotti, Mirko
Date :	23 November 2017
DOI :	10.3324/haematol.2017.178327
Copyright Disclaimer :	Copyright© 2018 Ferrata Storti Foundation Material published in Haematologica is covered by copyright. All rights are reserved to the Ferrata Storti Foundation. Use of published material is allowed under the following terms and conditions: https://creativecommons.org/licenses/by-nc/4.0/legalcode. Copies of published material are allowed for personal or internal use. Sharing published material for non-commercial purposes is subject to the following conditions: https://creativecommons.org/licenses/by-nc/4.0/legalcode, sect. 3. Reproducing and sharing published material for commercial purposes is not allowed without permission in writing from the publisher.
Depositing User :	Clive Harris
Date Deposited :	16 Nov 2017 15:04
Last Modified :	16 Jan 2019 19:04
URI:	http://epubs.surrey.ac.uk/id/eprint/844939

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