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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.

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Abstract

<p>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.</p> <p>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%).</p> <p>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.</p>

Item Type: Article
Divisions : Faculty of Health and Medical Sciences > School of Biosciences and Medicine
Authors :
NameEmailORCID
Donadon, IrvingUNSPECIFIEDUNSPECIFIED
McVey, Johnj.mcvey@surrey.ac.ukUNSPECIFIED
Garagiola, IsabellaUNSPECIFIEDUNSPECIFIED
Branchini, AlessioUNSPECIFIEDUNSPECIFIED
Mortarino, MimosaUNSPECIFIEDUNSPECIFIED
Peyvandi, FloraUNSPECIFIEDUNSPECIFIED
Bernardi, FrancescoUNSPECIFIEDUNSPECIFIED
Pinotti, MirkoUNSPECIFIEDUNSPECIFIED
Date : 2017
Copyright Disclaimer : © 2017 the authors
Depositing User : Clive Harris
Date Deposited : 16 Nov 2017 15:04
Last Modified : 16 Nov 2017 15:04
URI: http://epubs.surrey.ac.uk/id/eprint/844939

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