F VIII immunogenicity - Dr. H. Singer

In about 20-30% of patients with severe Haemophilia A (HA), treatment with replacement FVIII is complicated due to the development of inhibitory antibodies against the substituted concentrates. The mutation type and position in the protein plays a pivotal role in the risk of inhibitor development. It is believed that F8 nonsense null mutations provoke higher immunogenicity against replacement FVIII by lacking self-FVIII protein that could be presented to the immune system. However, surprisingly, non- sense mutations located in the light chain (A3C1C2 domains; last 1/3 of the protein), have higher risk to develop inhibitors when compared to nonsense mutations located in the heavy chain (A1A2B do- mains; first 2/3 part of protein). Accordingly, the highest inhibitor risk (70 %) appears, for two pre-terminal stop codons (PTCs) located in the A3 domain. This is against expectation as the protein should be largely available to get presented and recognized as self-protein. Molecular mechanism to explain this phenomenon is still lacking, however it was suggested that a rapid degradation due to the stop mutation could be responsible. To study the most native fate of FVIII mRNA and protein our group is generating induced pluripotent stem cells (iPSCs) from HA-patients.

Differentiation of IPS cells into vascular endothelial cells offers us a patient-specific cellular model to study intracellular fate of F8 mRNA and protein. Preliminary data of IPS differentiated vECs from HA-patients with different null mutations confirmed the existence of intracellular FVIII protein even if truncated. Therefor our major aim in our ongoing research is to identify the exact trafficking route of such truncated proteins (secretory or degradative pathway) and to find out if peptides from endogenous FVIII variants are presented by MHC-I and MHC-II on the surface of vascular endothelial cells. If so, we expect mutation-specific changes in the collection of MHC-presented peptides, that might have an effect on the inhibitor development associated with the specific mutation of a patient.

Dr. Heike Singer (Group Leader)
Payal Chawla (PhD student on EDUC8 ITN)
Melanie Rath (Technical assistant)

BHAP2017: EARLY CAREER INVESTIGATOR AWARD (Bayer)
GLEA2018: Günther Landbeck Excellence Award
EDUC8: Early Stage Researchers EDUCational Program on Factor VIII Immunogenicity. (Marie Skłodowska-Curie Actions (MSCA) Innovative Training Networks (ITN) H2020-MSCA-ITN-2019)

1) Jamil M.*, Singer H.*, Al-Rifai R., Nüsgen N., Rath M., Strauss S., Andreou I., Oldenburg J., El-Maarri O. Molecular analysis of fetal and adult primary human liver sinusoidal endothelial cells: a comparison to other endothelial cells. Int. J. Mol. Sci 2020 (PEER REVIEW)
2) Pezeshkpoor B, Gazorpak M, Berkemeier AC, Singer H, Pavlova A, Biswas A, Oldenburg J. In silico and in vitro evaluation of the impact of mutations in non-severe haemophilia A patients on assay discrepancies. Ann Hematol. 2019 Aug;98(8):1855-1865. doi: 10.1007/s00277-019-03691-1. Epub 2019 Apr 17.
3) Singer H, Biswas A, Zimmer N, Messaed C, Oldenburg J, Slim R, El-Maarri O. NLRP7 inter- domain interactions: the NACHT-associated domain is the physical mediator for oligomeric assembly. Mol Hum Reprod. 2014 Oct;20(10):990-1001.
4) Singer H, Walier M, Nüsgen N, Meesters C, Schreiner F, Woelfle J, Fimmers R, Wienker T, Kalscheuer VM, Becker T, Schwaab R, Oldenburg J, El-Maarri O. Methylation of L1Hs promoters is lower on the inactive X, has a tendency of being higher on autosomes in smaller genomes and shows inter-individual variability at some loci. Hum Mol Genet. 2012 Jan 1;21(1):219-35.
5) Singer H, Nüsgen N, El-Maarri O. SIRPH: an HPLC-based SNuPE for quantitative methylation measurement at specific CpG sites. Methods Mol Biol. 2011;791:89-100.
6) El-Maarri O, Walier M, Behne F, van Üüm J, Singer H, Diaz-Lacava A, Nüsgen N, Niemann B, Watzka M, Reinsberg J, van der Ven H, Wienker T, Stoffel-Wagner B, Schwaab R, Oldenburg J. Methylation at global LINE-1 repeats in human blood are affected by gender but not by age or natural hormone cycles. PLoS One. 2011 Jan 19;6(1):e16252.
7) El-Maarri O, Singer H, Klein C, Watzka M, Herbiniaux U, Brackmann HH, Schröder J, Graw J, Müller CR, Schramm W, Schwaab R, Haaf T, Hanfland P, Oldenburg J. Lack of F8 mRNA: a novel mechanism leading to hemophilia A. Blood. 2006 Apr 1;107(7):2759-65.

Stand: 12.10.2020