Variable readthrough responsiveness of nonsense mutations in hemophilia A
Martorell Cedres, Lluis (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Cortina, Vicente (Hospital Universitari Vall d'Hebron)
Parra, Rafael (Hospital Universitari Vall d'Hebron)
Barquinero, Jordi (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Vidal, Francisco (Banc de Sang i Teixits)
Universitat Autònoma de Barcelona

Data:	2020
Resum:	Readthrough therapy relies on the use of small molecules that enable premature termination codons in mRNA open reading frames to be misinterpreted by the translation machinery, thus allowing the generation of full-length, potentially functional proteins from mRNA carrying nonsense mutations. In patients with hemophilia A, nonsense mutations potentially sensitive to readthrough agents represent approximately 16% of the point mutations. The aim of this study was to measure the readthrough effect of different compounds and to analyze the influence of premature termination codon context in selected nonsense mutations causing hemophilia A. To this end, primary fibroblasts from three patients with hemophilia A caused by nonsense mutations (p. W1586X, p. Q1636X and p. R1960X) and Chinese hamster ovary (CHO) cells transfected with 12 different plasmids encoding mutated F8 (p. Q462X, p. Q1705X, p. Q1764X, p. W274X, p. W1726X, p. W2015X, p. W2131X, p. R1715X, p. R1822X, p. R1960X, p. R2071X and p. R2228X) were treated with gentamicin, geneticin, PTC124, RTC13 or RTC14. Responses were assessed by analyzing not only F8 mRNA expression and FVIII biosynthesis (FVIII antigen by ELISA, western blot and immunofluorescence) but also the FVIII activity (by chromogenic assay). In the patients' fibroblasts, readthrough agents neither stabilized F8 mRNA nor increased FVIII protein or activity to detectable levels. In CHO cells, only in five of the 12 F8 variants, readthrough treatment increased both FVIII antigen and activity levels, which was associated with a reduction in intracellular accumulation of truncated forms and an increase in full-length proteins. These results provide experimental evidence of genetic context dependence of nonsense suppression by readthrough agents and of factors predicting responsiveness.
Ajuts:	Instituto de Salud Carlos III PI11/03024 Instituto de Salud Carlos III PI11/0302
Drets:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, sempre que no sigui amb finalitats comercials, i sempre que es reconegui l'autoria de l'obra original.
Llengua:	Anglès
Document:	Article ; recerca ; Versió publicada
Publicat a:	Haematologica, Vol. 105 (february 2020) , p. 508-518, ISSN 1592-8721

DOI: 10.3324/haematol.2018.212118
PMID: 31197069

11 p, 5.5 MB

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