Abstract Number: PB0847
Meeting: ISTH 2020 Congress
Background: Development of inhibitory antibodies against FVIII are a great burden in the treatment of Hemophilia A (HA). F8 null mutations, like nonsense mutations, have a higher risk for inhibitor formation than other mutation types. The location of a specific nonsense mutation influences the risk for inhibitor development. F8 nonsense mutations located in a region coding for the FVIII heavy chain (A1-A2) have a lower risk to develop inhibitors, while F8 mutations located in the FVIII light chain (A3-C1-C2) have higher risk. By knowing this we generated IPS cells from ten severe HA-patients with different null mutations and differentiated the cells into vascular endothelial cells (vEC).
Aims: Our aim is to quantify F8 mRNA and intracellular FVIII protein in our patient-specific IPS derived vECs in order to understand the mutation-specific effect on inhibitor development.
Methods: Overlapping RT-PCR and Illumina human ht12-v4 expression array were used to analyze F8 mRNA expression in IPS differentiated vECs. Intracellular FVIII protein was quantified in wild type (Cm) and four patient vEC samples (L705X [StopA2], R1941X [StopA3], P-I22I [intron 22 inversion], P-LDA2 [Deletion Ex7-9]) using ELISA with different FVIII mAbs (GMA012/A2, GMA8011/LC, GMA8018/C1). Reference curves were prepared in the same sample lysis buffer.
Results: Full-length F8 mRNA was amplifiable for patients with nonsense mutation, showing highest mRNA expression for Cm and R1941X. Intracellular FVIII protein is detectable for Cm1 and P-I22I (20 mU/ml) when using mAbs GMA8018 and GMA8011. R1941X is only detectable with GMA8018 (10 mU/ml). P-LDA2 and L705X indicate an absence of protein.
Conclusions: We are able to quantify mU of intracellular FVIII in wild type and two HA-patient derived vEC samples. Absence of C1-specific ELISA signal in high-risk inhibitor mutation R1941X might indicate the existence of truncated protein.
To cite this abstract in AMA style:Singer H, Czogalla KJ, Rath M, Al-Rifai R, Jamil M, El-Maarri O, Oldenburg J. ELISA-based Quantification of intracellular FVIII Protein in IPS Derived Vascular Endothelial Cells from HA-patients with Nonsense Mutations [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/elisa-based-quantification-of-intracellular-fviii-protein-in-ips-derived-vascular-endothelial-cells-from-ha-patients-with-nonsense-mutations/. Accessed January 23, 2022.
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