Zeitschrift für Rheumatologie – Supplement 1 - Deutsche ...

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Abstracts
FVFER1 8. Forum Experimentelle Rheumatologie
Teil 1: Genetics of Autoimmunity
FVFER1-1
COMP-assoziierte Chondrodysplasien sind keine Speicherkrankheit
des endoplasmatischen Retikulums
Dinser R. 1 , Weirich C. 1 , Schmitz A. 2 , M U. 1 , Paulsson M. 2 , Zaucke F. 2
1 Klinik für Innere Medizin und Rheumatologie, Justus-Liebig Universität
Gießen, Kerckhoff -Klinik, 2 Zentrum für Biochemie, Medizinische Fakultät,
Universität zu Köln
Einleitung: Chondrodysplasien mit Assoziation zu Mutationen des
Cartilage Oligomeric Matrix Protein (COMP) sind durch disproportionierten
Minderwuchs, schwere früh einsetzende Arthrose und Instabilität
von Bändern und Sehnen charakterisiert. Bisher wurde die Pseudoachondroplasie
(PSACH) als schwerste Form als Speicherkrankheit
des endoplasmatischen Retikulums aufgefasst, da mutiertes COMP in
lamellären Einschlüssen des endoplasmatischen Retikulums von Knorpelzellen
aus Patientenbiopsien akkumuliert. Um die Pathophysiologie
dieser Erkrankung zu untersuchen, haben wir ein Zellkulturmodell
mittels adenoviralem Gentransfer entwickelt.
Methodik: Wir verglichen drei PSACH-assoziierte COMP Mutationen,
D475N, D469d, H587R durch Expression in primären Chondrozyten
und Tenozyten mittels adenoviraler Transduktion. Für Langzeitanalysen
wurden Chondrozyten in Alginat eingebettet.
Ergebnisse: Mutation H587R wird vergleichbar zum wildtyp-Protein
sezerniert, während die Mutationen D475N und D469d wie erwartet
im endoplasmatischen Retikulum akkumulieren. Alle drei Mutationen
verursachen schwere Fehlbildungen der in Kultur geformten Matrix
sowie apoptotischen Zelltod unabhängig von ihrem Sekretionsverhalten
und der untersuchten Zellart. Die Mutationen D475N und D469d
induzieren eine „unfolded protein response“ im Gegensatz zu H587R.
Schlussfolgerung: PSACH ist mehr als eine Speicherkrankheit des endoplasmatischen
Retikulum, da selbst unverzögert sezernierte COMP-
Mutationen eine Störung der Extrazellulärmatrix von Chondrozyten
und Tenozyten verursachen sowie apoptotischen Zelltod induzieren.
Vermutlich ist daher der Hauptfaktor der Pathogenese ein dominant negativer
Eff ekt von mutiertem COMP auf die Kollagen-Fibrillogenese.
FVFER1-2
Collagen type II recognition by an arthritogenic murine autoantibody:
no role of somatic mutation for high affi nity binding
Sehnert B. 2 , Böiers U. 2 , Päßler S. 2 , Lanig H. 3 , Holmdahl R. 4 , Burkhardt1 1 Abteilung Rheumatologie, Johann Wolfgang Goethe-Universität Frankfurt
am Main, 2 Medizinische Klinik III, Universität Erlangen–Nürnberg,
3 4 Computer-Chemie-Zentrum, Universität Erlangen–Nürnberg, Lund
University, Sweden
Objectives: Th e aim of the present study was to characterize the interaction
sites between the prototypic arthritogenic murine IgG mAB
CIIC1that is highly somatically mutated and its epitope on type II collagen
(CII, aa 359-369).
Methods: Th e establishment of a dynamic simulation modelling of a
CIIC1 single-chain fragment (scFV) in complex with the triple helical
CIIC1 epitope permitted structural insights into immune complex formation.
Th e computer-based data were experimentally tested by mutations
of predicted critical residues into alanine in the C1scFvs and
the respective CIIC1 epitope that were produced as recombinant constructs.
Th e binding affi nities of the mutated scFVs were determined by
ELISA and surface plasmon resonance measurements.
Results: Th e mutation experiments confi rmed the predicted interaction
sites of CII in the CDR2 heavy chain and CDR3 regions of both
heavy and light chain. Surprinsingly also the model prediction, that the
conversion of the C1scFv sequence into the respective germline does
| Zeitschrift für Rheumatologie · Supplement 1 · 2006
not aff ect CII binding affi nity (KD 3x 10-8) could be confi rmed experimentally
by the mutagenesis of 13(!) positions.
Conclusions: Our data indicate that potentially harmful cartilage specifi
c humoral autoimmunity is germline encoded. Th e molecular modeling
further demonstrate that the rigid collagen triple helix restricts
the likelihood of molecular interactions with the corresponding CDR
regions of the antibody considerably compared to globular antigens.
Th ese sterical constraints might provide an explanation why somatic
mutations have no obvious impact on CII recognition by the arthritogenic
autoantibody. Moreover, the structural insights into CII-autoantibody
interaction might be useful in future developments of collagenomimetic
ligands for therapeutic and diagnosistic purposes.
FVFER1-3
A polymorphism in the 3´UTR of the TNFRII gene is associated with
rheumatoid arthritis
Schulz A., Pierer M., Kaltenhäuser S., Arnold S., Seidel W., Häntzschel H.,
Wagner U.
Department of Medicine IV, University of Leipzig
Th e gene for tumor necrosis factor receptor II (TNFRII) is located on
chromoson 1p36.2, a region, which has already been described to be
associated with autoimmune diseases.
In the study presented here, a possible association of the following six
TNFRII polymorphisms with rheumatoid arthritis (RA) was analyzed:
two polymorphisms in exon 6 (gb:M32315: 676 T/G, 783 G/A), which
result in amino acid changes (Met196Arg and Glu232Lys); one polymorphism
in exon 2 (257 A/G); and three polymorphism in exon 10
(1663 G/A, 1668 T/G, 1690 T/C), which are located in the 3´UTR.
To identify these polymorphisms, polymerase chain reaction based
assays with sequence specifi c primers were used. Genomic DNA of
358 patients with RA according to the 1987 ACR criteria and 351 healthy
controls was genotyped.
Th e 1668 G allele in the 3´UTR (exon 10) was found more frequently in
RA patients than in healthy controls (5.45% vs. 2.71%, odds ratio= 2.07,
p=0.022).
For the 676 T/G polymorphism in exon 6, only a non-signifi cant trend
towards a higher frequency in RA patients was found. All other examined
polymorphisms were not associated with RA.
Th e results suggest that a polymorphism in the 3´UTR of the TNFRII
gene is associated with RA.
FVFER1-4
Analysis of mutations/SNPs in jun and fos proto-oncogene promoters
in the rheumatoid arthritis synovial membrane
Huber R. 1 , Lanick B. 1 , Ukena B. 1 , Dunger S. 2 , Pohlers D. 1 , Kinne RW. 1
1 Experimental Rheumatology Unit, Department of Orthopedics, Friedrich-
Schiller-University Jena, Waldkrankenhaus, 2 Franz-Volhard Clinic, Department
of Cardiology, HELIOS Klinikum-Berlin, Charité Campus Berlin-Buch
Objective: To identify relevant mutations and single nucleotide polymorphisms
(SNPs) in the promoters of jun and fos proto-oncogenes
in the synovial membrane (SM) of rheumatoid arthritis (RA), osteoarthritis
(OA), and normal control (NC) samples.
Methods: Mutations/SNPs in the respective jun/fos promoters were
identifi ed in DNA samples isolated from RA (n=10), OA (n=10), and
NC (n=5) SM tissue and blood by Non-Isotopic RNase Cleavage Assay.
Wildtype (wt) and mutated promoter fragments were cloned into
the reporter gene expression vector pUBT-luc (expressing luciferase).
Reporter gene expression of the respective promoter fragments was
measured in NIH 3T3 cells one and two days aft er transfection (both
constitutively and following stimulation with PMA) using a dual luciferase
assay system.