AG Prof. Dr. J. Oldenburg

Der Forschungsschwerpunkt unserer Arbeitsgruppe liegt bei den Enzymen des Vitamin K Zyklus (VKORC1 und GGCX) und den Vitamin K abhängigen Proteinen. Neben diesen bildet die Untersuchung heriditärer Störungen des Gerinnungssystems, insbesondere Defekte im Faktor VIII und IX (Hämophilie A und B), im Faktor XIII sowie im VWF die Grundlage weiterer wichtiger Projekte.
Unsere Forschungsaktivitäten werden durch Förderung seitens des BMBF (Nationales Genom Forschungs-Netzwerk- Cardiovaskuläre Erkrankungen), der DFG, der Universitätsklinik Bonn (BONFOR Programm) und der Industrie unterstützt.

Vitamin K cycle
F VIII immunogenicityPhenotype-Genotype Correlation in blood coagulation disordersFaktor-XIII-GenetiGenotype-phenotype correlation in von Willebrand disease

Vitamin K-cycle

The role of vitamin K beyond coagulation
Vitamin K is essential to drive a post-translational modification, called γ-carboxylation, of 15 different vitamin K dependent proteins including several clotting factors but also non-haemostatic proteins such as GRP, MGP, Osteocalcin, PRGP1 and 2, TMG3 and 4 and Gas6. These vitamin K dependent non-haemostatic proteins have function in calcification, cell-signalling and other not yet identified pathways.

Our Emmy Noether research group study the function of the vitamin K cycle beyond blood coagulation and the interplay of the enzymes VKORC1, VKORC1-like1, and GGCX with vitamin K dependent proteins in cellular and mice models under heathy and disease conditions.

Group members: Dr. rer. nat. Katrin Czogalla-Nitsche, PhD
Emmy-Noether group leader / Principle investigator
Room: 2G/302
Phone: +49 (0)228-287 16783
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Prof. Dr. med. Johannes Oldenburg
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Suvoshree Ghosh
PhD student
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Francesco Forin
PhD student
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Anna-Lena Buhl
PhD student
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Katrin Kraus
Technician (BTA)
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Heike Höfer
Technican (BTA)
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Publications: Czogalla KJ, Liphardt K, Höning K, Hornung V, Biswas A, Watzka M, Oldenburg J.; VKORC1 and VKORC1L1 have distinctly different oral anticoagulant dose-response characteristics and binding sites.; Blood Adv. 2018 Mar;27;2(6):691-702.
Czogalla KJ, Biswas A, Höning K, Hornung V, Liphardt K, Watzka M, Oldenburg J.; Warfarin and vitamin K compete for binding to Phe55 in human VKOR.; Nat Struct Mol Biol. 2017 Jan;24(1):77-85.
Czogalla KJ, Biswas A, Rost S, Watzka M, Oldenburg J.; The Arg98Trp mutation in human VKORC1 causing VKCFD2 disrupts a di-Arginine-based ER retention motif.; Blood. 2014 Aug 21;124(8):1354-62
Czogalla KJ, Biswas A, Wendeln AC, Westhofen P, Müller CR, Watzka M, Oldenburg J.; Human VKORC1 mutations cause variable degrees of 4-hydroxycoumarin resistance and affect putative warfarin binding interfaces.; Blood. 2013 Oct 10;122(15):2743-50.

Stand: 04. September 2020

F VIII immunogenicity

Dr. Heike Singer (Junior Research Group Leader)

Staff:
Payal Chawla (PhD student on EDUC8 ITN)
Melanie Rath (Technical assistant)

Research area: FVIII immunogenicity

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.

Funding:
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)

Publications:
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.
5) 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

Phenotype-Genotype Correlation in blood coagulation disorders
In the department of molecular haemostaseology 25 genes involved in blood coagulation are routinely investigated. The scientific interests of the group are focused on genotype-phenotype correlation in patients with rare blood coagulation disorders. Haemophilia, a deficiency of coagulation factor VIII and IX is of special interest. Different genetic defects have been identified and functionally studied. New insights in the genetic alteration as deep intronic changes leading to haemophilia are thoroughly explored. An interesting issue addressed in our group is the association F8 missense mutations and their impact on the FVIII activity, especially in the context of discrepancy between one stage clotting and chromogenic assays. Inhibitor development, is of special interest and different genetic factors as mutation type, polymorphisms in the immune-response genes and HLA are currently investigated. Another scientific area focuses on factor 5 gene. New mutations, leading to APC resistance have been identified and structurally and functionally studied.
The group is involved in a number of national and international studies including ABIRISK, analyzing the mechanisms and consequences of immunization against biopharmaceutical products and OBSITI aiming to evaluate patient and therapy related variables on immune tolerance induction course.

Working group:
Priv.-Doz. Dr. Anna Pavlova, MD, PhD
Dr. Behnaz Pezeshkpoor, PhD
Dr. Thilo Albert, PhD

Selection of publications:
1. Goodeve AC, Pavlova A, Oldenburg J. Genomics of bleeding disorders. Haemophilia. 2014 May;20 Suppl 4:50-3.
2. Pavlova A, Delev D, Pezeshkpoor B, Müller J, Oldenburg J. Haemophilia A mutations in patients with non-severe phenotype associated with a discrepancy between one-stage and chromogenic factor VIII activity assays. Thromb Haemost.2014 May 5;111(5):851-61.
3. Pahl S, Pavlova A, Driesen J, Oldenburg J. Effect of F8 B domain gene variants on synthesis, secretion, activity and stability of factor VIII protein. Thromb Haemost. 2014 Jan;111(1):58-66.
4. Pezeshkpoor B, Pavlova A, Oldenburg J, El-Maarri O. F8 genetic analysis strategies when standard approaches fail. Hamostaseologie. 2014;34(2):167-73.
5. Pavlova A, Oldenburg J. Defining severity of hemophilia: more than factor levels. Semin Thromb Hemost. 2013 Oct;39(7):702-10. doi: 10.1055/s-0033-1354426. Epub 2013 Sep 11. Review.
6. Pavlova A. F8 gene and phenotype: single player in a team? Blood. 2013 May,9;121(19):3784-5.
7. Pahl S, Pavlova A, Driesen J, Müller J, Pötzsch B, Oldenburg J. In vitro characterization of recombinant factor VIII concentrates reveals significant differences in protein content, activity and thrombin activation profile.Haemophilia. 2013 May;19(3):392-8.
8. Schwaab R, Pavlova A, Albert T, Caspers M, Oldenburg J. Significance of F8 missense mutations with respect to inhibitor formation. Thromb Haemost. 2013 Mar;109(3):464-70. doi: 10.1160/TH12-07-0521. Epub 2013 Jan 10. PubMed PMID: 23306409
9. Caspers M, Pavlova A, Driesen J, Harbrecht U, Klamroth R, Kadar J, Fischer R,Kemkes-Matthes B, Oldenburg J. Deficiencies of antithrombin, protein C andprotein S - practical experience in genetic analysis of a large patient cohort.Thromb Haemost. 2012 Aug;108(2):247-57.
10. Luxembourg B, Delev D, Geisen C, Spannagl M, Krause M, Miesbach W, Heller C, Bergmann F, Schmeink U, Grossmann R, Lindhoff-Last E, Seifried E, Oldenburg J,Pavlova A. Molecular basis of antithrombin deficiency. Thromb Haemost. 2011Apr;105(4):635-46.
11. Pavlova A, Delev D, Lacroix-Desmazes S, Schwaab R, Mende M, Fimmers R, Astermark J, Oldenburg J. Impact of polymorphisms of the major histocompatibility complex class II, interleukin-10, tumor necrosis factor-alpha and cytotoxic T-lymphocyte antigen-4 genes on inhibitor development in severe hemophilia A. J Thromb Haemost. 2009 Dec;7(12):2006-2015. doi: 10.1111/j.1538-7836.2009.03636.x. PubMed PMID: 19817985.

Stand: 02. Dezember 2014

Faktor-XIII-Genetik
Plasma Factor XIII is a protransglutaminase circulating as a heterotetramer composed of two catalytic A and two protective B subunits. It functions by crosslinking preformed fibrin clots and also by crosslinking fibrinolytic inhibitors like alpha 2-antiplasmin to the fibrin clot making the clot mechanically and chemically stronger and resistant to premature fibrinolysis. Deficiency of this protein can result in a bleeding predisposition commonly known as Factor XIII deficiency. Factor XIII deficiency can be inherited or acquired. The inherited form is of two types: A) a rare severe form (1 in 1 to 4 million prevalence and B) a more frequent mild heterozygous form. The inherited form of this deficiency is characterized by mutations occurring in the F13A1 and F13B genes; the genes for the A and B subunits respectively.
The Factor XIII group works on two aspects: A) The genotypic and phenotypic diagnosis of patients suffering from severe and mild Factor XIII deficiency and B) The structure-functional aspects of Factor XIII. The Factor XIII group has in the past characterized and reported a large number of Factor XIII deficiency patients with mutations in F13A and F13B genes. The efforts of the Factor XIII group in the last few years have brought the otherwise lesser known mild Factor XIII deficiency into focus. This group also is one of the few groups in the world that is involved in determining the molecular etiology of heterozygous mutations reported from mild Factor XIII deficiency patients. Current efforts include expressing these mutations in heterologous systems and evaluating the in vitro expression phenotype in a multitude of assays each phenotypically representing a unique aspect of Factor XIII. Current efforts are also directed towards solving the Factor XIII A2B2 heterotetramer structure and eventually characterizing it in terms of complex dynamics.

Working group:
Dr. (biol.) Arijit Biswas, Scientist
Priv.-Doz. Dr. (med.) Vytautas Ivaskevicius, Clinician
Ms. Sneha Gupta, Ph.D student

Recent publications from this group are:

Biswas A, Ivaskevicius V, Thomas A, Varvenne M, Brand B, Rott H, Haussels I, Ruehl H, Scholz U, Klamroth R, Oldenburg J. Eight novel F13A1 gene missense mutations in patients with mild FXIII deficiency: in silico analysis suggests changes in FXIII-A subunit structure/function. Ann Hematol. 2014 Jun 3.

Souri M, Biswas A, Misawa M, Omura H, Ichinose A. Severe congenital Factor XIII deficiency caused by novel W187X and G273V mutations in the F13A gene; diagnosis and classification according to the ISTH/SSC guidelines. Haemophilia. 2014 Mar;20(2):255-62.

Biswas A, Ivaskevicius V, Thomas A, Oldenburg J. Coagulation factor XIII deficiency. Diagnosis, prevalence and management of inherited and acquired forms. Hamostaseologie. 2014;34(2):160-6.

Biswas A, Thomas A, Bevans CG, Ivaskevicius V, Oldenburg J. In vitro secretion deficits are common among human coagulation factor XIII subunit B missense mutants: Correlations with patient phenotypes and molecular models. Hum Mutat. 2013 Aug 2.

Ivaskevicius V, Biswas A, Thomas A, Lyonga S, Rott H, Halimeh S, Kappert G, Klammroth R, Scholz U, Eberl W, Harbrecht U, Gnida C, Hertfelder HJ, Marquardt N, Oldenburg J. A common F13A1 intron 1 variant IVS1+12(A) is associated with mild FXIII deficiency in Caucasian population. Ann Hematol. 2013 Jul;92(7):975-9.

Biswas A, Ivaskevicius V, Seitz R, Thomas A, Oldenburg J. An update of the mutation profile of Factor 13 A and B genes. Blood Rev. 2011 Sep;25(5):193-204.

Ivaskevicius V, Biswas A, Bevans C, Schroeder V, Kohler H.P, Rott H, Halimeh S, Petrides P.E, Lenk H, Krause M, Miterski B, Harbrecht U, Oldenburg J. Identification of eight novel coagulation Factor XIII subunit A mutations: Implied consequences for structure and function. Haematologica. 2010 Feb 23.

Ivaskevicius V, Biswas A, Loreth R, Schroeder V, Ohlenforst S, Rott H, Krause M, Kohler H.P, Scharrer I, Oldenburg J. Mutations affecting disulphide bonds contribute to a fairly common prevalence of F13B gene defects: Results of a genetic study in 14 families with Factor XIII B deficiency. Haemophilia. 2010 Jul 1; 16(4):675-82.

Stand: 02. Dezember 2014

Genotype-phenotype correlation in von Willebrand disease
Von Willebrand disease (VWD) project is focused on understanding of the molecular basis of different types of VWD to establish phenotype-genotype correlations.
In VWD project, we explore genotype and phenotype characteristics of a cohort of patients with VWD with the aim of dissecting the distribution of mutations in different types of VWD and correlate them to the clinical disease severity. In addition, our project intends to elucidate the pathophysiological mechanisms of detected novel missense and potential splice mutations by in vitro gene expression studies in mammalian cell lines and in vivo transcript analysis respectively. Moreover, possible structural impact of the mutations on VWF protein is studied by in silico homology modeling. Additionally, we plan to study behavior of the recombinant wilde-type and mutant VWF strings in vitro under shear-stress conditions resembling the blood flow in vessels using an automated shear-controlling device.

Working group
Dr. Hamideh Yadegari, PhD

Publications

Yadegari H, Driesen J, Pavlova A, Biswas A, Ivaskevicius V, Klamroth R, Oldenburg J. Insights into pathological mechanisms of missense mutations in C-terminal domains of von Willebrand factor causing qualitative or quantitative von Willebrand disease. Haematologica. 2013 Aug;98(8):1315-23. doi: 10.3324/haematol.2013.084111. Epub 2013 Mar 28. PubMed PMID: 23539537; PubMed Central PMCID: PMC3729914.

Yadegari H, Driesen J, Pavlova A, Biswas A, Hertfelder HJ, Oldenburg J. Mutation distribution in the von Willebrand factor gene related to the different von Willebrand disease (VWD) types in a cohort of VWD patients. Thromb Haemost. 2012 Oct;108(4):662-71. Epub 2012 Aug 7. PubMed PMID: 22871923.

Yadegari H, Driesen J, Hass M, Budde U, Pavlova A, Oldenburg J. Large deletions identified in patients with von Willebrand disease using multiple ligation-dependent probe amplification. J Thromb Haemost. 2011 May;9(5):1083-6. doi: 10.1111/j.1538-7836.2011.04260.x. PubMed PMID: 21410641.

Stand: 02. Dezember 2014