Page 274 - 2020_02-Haematologica-web

Coagulation & its Disorders
Ferrata Storti Foundation
Haematologica 2020 Volume 105(2):508-518
Variable readthrough responsiveness of nonsense mutations in hemophilia A
Lluis Martorell,1,2 Vicente Cortina,3 Rafael Parra,4 Jordi Barquinero1,* and Francisco Vidal2,5,6*
1Gene and Cell Therapy Laboratory, Vall d'Hebron Institut de Recerca (VHIR), Universitat Autònoma de Barcelona (UAB); 2Congenital Coagulopathies Laboratory, Banc de Sang i Teixits (BST); 3Vall d'Hebron Core Laboratory (Section of Thrombosis and Haemostasis), Hospital Vall d’Hebron; 4Banc de Sang i Teixits-Hospital Vall d’Hebron; 5Molecular Diagnosis and Therapy Unit, VHIR-UAB and 6CIBER de Enfermedades Cardiovasculares (CIBERCV), Barcelona, Spain
*JB and FV contributed equally to this work as co-senior authors.
ABSTRACT
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 gen- eration 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 hemo- philia A. To this end, primary fibroblasts from three patients with hemo- philia A caused by nonsense mutations (p.W1586X, p.Q1636X and p.R1960X) and Chinese hamster ovary (CHO) cells transfected with 12 dif- ferent 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 analyz- ing 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 nei- ther 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 pro- vide experimental evidence of genetic context dependence of nonsense sup- pression by readthrough agents and of factors predicting responsiveness.
Introduction
Hemophilia A (HA) is an X-linked disorder caused by molecular defects in the coagulation factor VIII gene (F8). Nonsense mutations represent approximately 16% of point mutations leading HA (http://www.factorviii-db.org). These patients usually have severe HA and a high risk of FVIII inhibitor development.1 HA predis- poses patients to recurrent bleeds, primarily into the joints and soft tissues, with the severity of the disease inversely correlated to FVIII:C activity. Therapy is based on the prevention of bleeding using FVIII replacement (either recombinant or plas- ma-derived), with an annual estimated cost of >150,000 euros in severely affected patients.2
Although major advances have been reported with gene therapy in both HA3 and hemophilia B (HB),4 it has yet to be approved for clinical use and will not, therefore, be commercially available for the majority of patients in the immediate future. Recently, readthrough therapy has emerged as a potential strategy for the treat-
Correspondence:
JORDI BARQUINERO/FRANCISCO VIDAL PEREZ
jordi.barquinero@vhir.org/fvidal@bst.cat
Received: November 23, 2018. Accepted: June 11, 2019. Pre-published: June 13, 2019.
doi:10.3324/haematol.2018.212118
Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/105/2/508
©2020 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 inter- nal use. Sharing published material for non-commercial pur- poses is subject to the following conditions: https://creativecommons.org/licenses/by-nc/4.0/legalcode, sect. 3. Reproducing and sharing published material for com- mercial purposes is not allowed without permission in writing from the publisher.
508
haematologica | 2020; 105(2)
ARTICLE