Variable readthrough responsiveness of nonsense mutations in hemophilia A

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 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.

Figure 1.

F8 mutations studied and detection of F8 mRNA levels. (A) Schematic representation of the distribution of premature termination codons (PTC) across the F8BDD cDNA generated by site-directed mutagenesis for the Chinese hamster ovary (CHO) model or naturally occurring in the hemophilia A (HA) patients. The black arrow represents the F8 cDNA (5’ to 3’), numbers below the arrow correspond to the nucleotide position, while the gray bar represents the BDD-FVIII protein, and numbers below correspond to the amino acid position according to the Human Genome Variation Society (HGVS) nomenclature. The distribution of mutations in F8 mRNA analyzed in CHO model, patients’ fibroblasts (in gray boxes) or both cellular models (black lined gray box) are also shown. BD-L: BDD-linker; FVIII-HC: heavy chain; FVIII-LC: light chain. (B) F8 mRNA levels detected by quantitative real-time polymerase chain reaction in the fibroblasts of HA-patients or a normal control. GAPDH: glyceraldehyde 3-phosphate dehydrogenase; Control: fibroblasts of a HB patient; Q1636X, W1586X and R1960X HA patients fibroblasts harboring these nonsense mutations; and R1960Q: HA patient fibroblasts harboring this missense mutation. CT: untreated cells; GN: geneticin 100 mg/mL; GT: gentamicin 100 mg/mL; PTC: PTC124 10 mM; RTC13: RTC13 10 mM; CHX: cycloheximide 1 mg/mL (n=3). (C) Time course of F8BDD mRNA levels detected by BDD-specific semi-quantitative polymerase chain reaction in the CHO-based HA-model. F8BDD: WT variant; p.Q1705X, p.W1726X and p.R1960X: F8BDD variants harboring PTC; CHO: non transfected CHO cells; Huh-7: human hepatocellular carcinoma cell line (a cell line that expresses high levels of endogenous F8 but used here as a negative control of the F8BDD-specific amplification); MW: 100-bp DNA ladder. P values: ^*P<0.05, ^**P<0.01 and ^*** P<0.001. (n=3).

Figure 2.

Readthrough agents (RTA) can increase FVIII antigen levels in the supernatants of some transfected Chinese hamster ovary (CHO) cells. Human FVIII:Ag levels analyzed by ELISA in the supernatants of CHO cells transiently transfected with the F8 variants, showing the increase in FVIII:Ag levels before and after RTA-treatment versus the F8 wild-type (WT) variant. BDD-FVIII: WT; CT: untreated controls; GN100: 100 mg geneticin/mL and GT100: 100 mg gentamicin/mL. ^*P<0.05; ^**P<0.01; ^***P<0.001. (N=3).

Figure 3.

Gentamicin increased FVIII biosynthesis in some transfected Chinese hamster ovary (CHO) cells. Representative images of the immunofluorescence detection of FVIII in transiently transfected CHO cells stained with antibodies against either FVIII heavy chain or FVIII light chain (both in green). Gentamicin treatment (GT50: 50 mg/mL) increased FVIII labeling using the anti-FVIII light chain antibody in CHO cells transiently transfected with variants harboring the p.W274X, p.Q462X, p.Q1764X, p.W2015X and p.W2131X premature termination codons.

Figure 4.

Readthrough agents (RTA) reduced intracellular accumulation of FVIII in transfected Chinese hamster ovary (CHO) cells. Representative images of the immunodetection of FVIII heavy chain in cell homogenates of transiently transfected CHO cells. (A) Western blot of CHO cells transfected with either wild-type (WT) BDD-FVIII (166.19 kDa), the p.Q462X (52.8 kDa), p.Q1764X (98.51 kDa) (top panel), p.W2015X (127.9 kDa), or the p.R1822X (105.24 kDa) variants (bottom panel). The intracellular accumulation of BDD-FVIII or the truncated proteins before and after RTA-treatment is shown. (B) Densitometry of the bands in (A). CT: Untreated cells; GN50: 50 mg geneticin/mL; GN100: 100 mg geneticin/mL; GT50: 50 mg gentamicin/mL; GT100: 100 mg gentamicin/mL; PTC: PTC124 10 mM; RTC13 and RTC14: 10 mM; Mock: untransfected cells; MW: Pageruler Plus prestained protein ladder; Refacto®: recombinant human BDD-FVIII (Pfizer); LR: loading reference used for densitometry normalization.

Figure 5.

Readthrough agents (RTA) increase FVIII coagulant activity in the supernatants of transfected Chinese hamster ovary (CHO) cells. (A) Fold increase of FVIII:C (relative to untreated controls) in the supernatants of cultured CHO cells transiently transfected with variants harboring a QX premature termination codons (PTC) (see legend to Figure 1). (B) Fold increase of FVIII:C in the supernatants of cultured CHO cells transiently transfected with variants harboring a WX PTC (see legend to Figure 1). (C) Fold increase of FVIII:C in the supernatants of cultured CHO cells transiently transfected with variants harboring a RX PTC (see legend to Figure 1). CT: untreated controls; GN50: 50 mg geneticin/mL; GN100: 100 mg geneticin/mL; GT50: 50 μg gentamicin/mL; GT100: 100 mg gentamicin/mL; PTC: PTC124 10 mM; RTC13 and RTC14: 10 μM. *P<0.05; **P<0.01; ***P<0.001. (N=3).