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 U7 snRNA supported the presence of a splicing regulatory element, potentially affected by several missense mutations causing hemophilia A. 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 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 pat-terns produce a gradient of residual activity, and clarify genotype-pheno-type relationships. Data detail pathogenic mechanisms that, only in combination, account for moderate/severe disease forms, which in turn determine the mutation profile. Taken together we provide a clear exam-le of interplay between mRNA and protein mechanisms of disease that operate in shaping many other inherited disorders.

Clustered F8 missense mutations cause hemophilia A by combined alteration of splicing and protein biosynthesis and activity / I. Donadon, J.H. Mcvey, I. Garagiola, A. Branchini, M. Mortarino, F. Peyvandi, F. Bernardi, M. Pinotti. - In: HAEMATOLOGICA. - ISSN 0390-6078. - 103:2(2018 Feb), pp. 344-350.

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

I. Garagiola;M. Mortarino;F. Peyvandi;
2018-02

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 U7 snRNA supported the presence of a splicing regulatory element, potentially affected by several missense mutations causing hemophilia A. 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 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 pat-terns produce a gradient of residual activity, and clarify genotype-pheno-type relationships. Data detail pathogenic mechanisms that, only in combination, account for moderate/severe disease forms, which in turn determine the mutation profile. Taken together we provide a clear exam-le of interplay between mRNA and protein mechanisms of disease that operate in shaping many other inherited disorders.
hematology
Settore MED/09 - Medicina Interna
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/557165
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