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. 2019 Jun 13;105(2):508–518. doi: 10.3324/haematol.2018.212118

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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).