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Variable nonsense suppression in Hemophilia A
Table 1. Molecular and clinical data of patients with hemophilia A included in the study.
Patient Gene region
#1 Exon 14
#2 Exon 14
#3 Exon 18
#4 Exon 18
Nucleotide change
c.4757G>A
c.4906C>T
c.5878C>T
c.5879G>A
Amino acid change [HGVS]
p.W1586X
p.Q1636X
p.R1960X
p.R1960Q
Nucleotide context (-1Stop+4)
TTAGG
ATAAA
TTGAT
(missense)
Phenotype
Severe
Severe
Moderate
Mild
FVIII:C % Inhibitors
<1 Yes
<1 nd
4 No
10-15 No
FVIII:Ag %
nd
nd nd nd
FVIII:Ag: FVIII antigen levels; FVIII:C: Factor VIII coagulant activity; HGVS: Human Genome Variation Society nomenclature; nd: not determined.
ment of hereditary diseases caused by nonsense muta- tions. It is based on the use of small molecules that cause ribosomes to ignore premature termination codons (PTC) during translation, thus allowing the generation of full- length, potentially functional, proteins from the mutant mRNA. PTC are estimated to occur in approximately 10- 15% of the patients with monogenic disease (homo or heterozygotes), who may potentially benefit from readthrough agents (RTA). However, this approach has important limitations, including the toxicities of some of these compounds (which require long-term administra- tion), the usually very low levels of readthrough that can be obtained, and the fact that an amino acid different from that originally encoded may be introduced at the PTC site, with potentially detrimental consequences in terms of protein function. Nonetheless, clinical trials or pilot stud- ies of RTA have already been carried out in patients with cystic fibrosis,5,6 Duchenne muscular dystrophy7,8 or hemo- philia,9 although often with inconclusive results.
The aminoglycosides gentamicin and geneticin, as well as non-aminoglycoside compounds, such as PTC124 (Ataluren),10 RTC13 and RTC14,11 have been used as RTA based on their ability to suppress PTC and to allow the gen- eration of full-length proteins with at least partial function- ality. However, the efficiency of RTA therapy varies accord- ing to not only the PTC type and sequence context,12-14 but also to the specific agent that is used, which can influence the binding of specific aminoacidyl-tRNA to the PTC and thus the amino acid added to the nascent peptide, with con- sequences for the functionality of the recoded protein.15 Here, we present an analysis of the effect of five RTA (gen- tamicin, geneticin, PTC124, RTC13 and RTC14) on F8 mRNA expressed in primary skin fibroblasts from three patients with HA as well as in a Chinese hamster ovary (CHO)-cell-based model of HA. Our aim was to assess the readthrough effect of these RTA on the FVIII activity, in addition to FVIII:Ag levels, and the influence of the molec- ular context, including type of stop codon, adjacent sequences, and the amino acid originally encoded by the wild-type (WT) protein at the mutated site.
Methods
Patients and isolation of skin fibroblasts
Four patients with HA caused by either nonsense mutations (p.W1568X, p.Q1636X and p.R1960X) or a missense mutation (p.R1960Q), diagnosed at the Hemophilia Unit of the Vall d'Hebron University Hospital and genetically characterized at the Congenital Coagulopathies Laboratory of the Blood and Tissue Bank of Catalonia (BST)16 were selected for this study. All partici-
pating patients and controls provided informed consent in accor- dance with the Declaration of Helsinki. The study was approved by our institutional Research Ethics Committee.
The genetic characteristics of each patient and their plasma FVIII:C activities at the time of diagnosis are summarized in Table 1.
Generation of F8 variants harboring premature termination codon mutations
All F8 B-domain deleted (F8BDD) cDNA variants were designed according to our hypotheses and purchased from GeneArt (Thermo Fisher Scientific, Waltham, MA, USA). All the mutations studied are shown in Figure 1A.
Cell lines and Chinese hamster ovary-cellular model
Human hepatocarcinoma cell line Huh-7 was kindly provided by Dr. J. Quer (VHIR, Barcelona) while CHO-S cells were pur- chased from Thermo Fisher Scientific. Both cell lines were cul- tured in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum, penicillin and streptomycin (all from Biowest, Nuaillé, France). For CHO transfection, cells were passed three times and seeded at 4-5 x 104 cells in 12-well plates to achieve a confluence of 50-80%. The day after, the cells were transfected with 1.5 μg of the WT or mutated F8BDD plasmids using 1.5 μL lipofectamine-LTX (Thermo Fisher Scientific) per well, following the manufacturer’s recommendations.
Readthrough agent treatment
Patient-derived skin fibroblasts (3.5x103 cells/cm2) were seeded in 6-well plates and grown to 70-80% confluence before RTA treatment, while CHO cells were treated after 8 hours of transfec- tion. The cells were treated with gentamicin, geneticin (Thermo Fisher Scientific), PTC124 (Selleckchem Co., Houston, TX, USA), RTC13 or RTC14 (ID: 5735019 and ID: 5311257) (ChemBridge. San Diego, CA, USA) using a range of concentrations reported to induce PTC readthrough in vitro (50-100 μg/mL for gentamicin and geneticin, 10 μM for PTC124, RTC13 and RTC14).17,18
F8 mRNA analysis
Total RNA was extracted using the RNeasy mini kit followed
by on-column DNase I treatment (Qiagen. Hilden, Germany). Single-stranded cDNA was generated with the high capacity cDNA reverse transcription kit (Thermo Fisher Scientific) using 500 ng of total RNA and random primers in a final volume of 25 μL, as previously described.19 The cDNA obtained was used to quantify F8 mRNA expression.
FVIII Ag levels of F8BDD variants
Human FVIII antigen levels (FVIII:Ag) were determined in cul-
ture supernatants using the IMUBIND® factor VIII ELISA kit (Sekisui Diagnostics, Lexington, MA, USA).
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