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Oliver von Kampen,
Stephan Buch,
Michael Nothnagel,
Lorena Azocar,
Hector Molina,
Mario Brosch,
Wiebke Erhart,
Witigo von Schönfels,
Jan Egberts,
Marcus Seeger, [......],
Jürgen Tepel,
Klaus Puschel,
Thomas Becker,
Stefan Schreiber,
Matthias Platzer,
Henry Völzke,
Michael Krawczak,
Juan Francisco Miquel,
Clemens Schafmayer,
Jochen Hampe
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ABSTRACT: BACKGROUND & AIMS: The sterolin locus (ABCG5/ABCG8) has been solidly shown to confer susceptibility for cholesterol gallstone disease in humans. Both the responsible variant and the molecular mechanism causing an increased incidence of gallstones in these patients have as yet not been identified. METHODS: Genetic mapping utilized patient samples from Germany (2808 cases, 2089 controls), Chile (680 cases, 442 controls), Denmark (366 cases, 766 controls), India (247 cases, 224 controls) and China (280 cases, 244 controls). Analysis of allelic imbalance in cDNA samples from human liver (N=22) was performed using pyrosequencing. Transiently transfected HEK293 cells were used for [3H]-cholesterol export assays, analysis of protein expression and localisation of allelic constructs. RESULTS: Through fine mapping in German and Chilean samples, a ∼250kB disease-associated interval could be defined for this locus. Lack of allelic imbalance or allelic splicing of the ABCG5 and ABCG8 transcripts in human liver limited the search to coding SNPs. Subsequent mutation detection and genotyping yielded two disease-associated variants ABCG5-R50C (p=4.94×10(-9) ) and ABCG8-D19H (p=1.74×10(-10) ) in high pair-wise LD (r(2) =0.95). [3H]-cholesterol export assays of allelic constructs harbouring these genetic candidate variants demonstrated increased transport activity (3.2-fold, p=0.003) only for the ABCG8-19H variant, which was also superior in nested logistic regression models in German (p=0.018), Chilean (p=0.030) and Chinese (p=0.040) patient samples. CONCLUSION: This variant thus provides a molecular basis for biliary cholesterol hypersecretion as the mechanism for cholesterol gallstone formation thereby drawing a link between "post-genomic" and "pre-genomic" pathophysiological knowledge about this common complex disorder. (HEPATOLOGY 2012.).
Hepatology 08/2012; · 11.66 Impact Factor
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Ole Ammerpohl,
Johann Pratschke,
Clemens Schafmayer,
Andrea Haake,
Wladimir Faber, Oliver von Kampen,
Mario Brosch,
Bence Sipos,
Witigo von Schönfels,
Katharina Balschun, [......],
Alexander Arlt,
Bodo Schniewind,
Jonas Grauholm,
Holger Kalthoff,
Peter Neuhaus,
Felix Stickel,
Stefan Schreiber,
Thomas Becker,
Reiner Siebert,
Jochen Hampe
[show abstract]
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ABSTRACT: Abberrant DNA methylation is one of the hallmarks of cancerogenesis. Our study aims to delineate differential DNA methylation in cirrhosis and hepatic cancerogenesis. Patterns of methylation of 27,578 individual CpG loci in 12 hepatocellular carcinomas (HCCs), 15 cirrhotic controls and 12 normal liver samples were investigated using an array-based technology. A supervised principal component analysis (PCA) revealed 167 hypomethylated loci and 100 hypermethylated loci in cirrhosis and HCC as compared to normal controls. Thus, these loci show a "cirrhotic" methylation pattern that is maintained in HCC. In pairwise supervised PCAs between normal liver, cirrhosis and HCC, eight loci were significantly changed in all analyses differentiating the three groups (p < 0.0001). Of these, five loci showed highest methylation levels in HCC and lowest in control tissue (LOC55908, CELSR1, CRMP1, GNRH2, ALOX12 and ANGPTL7), whereas two loci showed the opposite direction of change (SPRR3 and TNFSF15). Genes hypermethylated between normal liver to cirrhosis, which maintain this methylation pattern during the development of HCC, are depleted for CpG islands, high CpG content promoters and polycomb repressive complex 2 (PRC2) targets in embryonic stem cells. In contrast, genes selectively hypermethylated in HCC as compared to nonmalignant samples showed an enrichment of CpG islands, high CpG content promoters and PRC2 target genes (p < 0.0001). Cirrhosis and HCC show distinct patterns of differential methylation with regards to promoter structure, PRC2 targets and CpG islands.
International Journal of Cancer 04/2011; 130(6):1319-28. · 5.44 Impact Factor
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Stephan Buch,
Clemens Schafmayer,
Henry Völzke,
Marcus Seeger,
Juan F Miquel,
Silvia C Sookoian,
Jan H Egberts,
Alexander Arlt,
Carlos J Pirola,
Markus M Lerch, [......],
Andre Franke, Oliver von Kampen,
Mario Brosch,
Michael Nothnagel,
Wolfgang Kratzer,
Bernhard O Boehm,
Dieter C Bröring,
Stefan Schreiber,
Michael Krawczak,
Jochen Hampe
[show abstract]
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ABSTRACT: Genome-wide association studies have mapped loci that are associated with serum levels of bilirubin. Bilirubin is a major component of gallstones so we investigated whether these variants predict gallstone bilirubin content and overall risk for gallstones.
Loci that were identified in a meta-analysis to attain a genome-wide significance level of a P value less than 1.0×10(-7) (UGT1A1, SLCO1B1, LST-3TM12, SLCO1A2) were analyzed in 1018 individuals with known gallstone composition. Gallstone risk was analyzed in 2606 German choleystecomized individuals and 1121 controls and was replicated in 210 cases and 496 controls from South America.
By using the presence of bilirubin as a phenotype, variants rs6742078 (UGT1A1; P = .003), rs4149056 (SLCO1B1; P = .003), and rs4149000 (SLCO1A2; P = .015) were associated with gallstone composition. In regression analyses, only UGT1A1 and SLCO1B1 were independently retained in the model. UGT1A1 (rs6742078; P = .018) was associated with overall gallstone risk. In a sex-stratified analysis, only male carriers of rs6742078 had an increased risk for gallstone disease (P = 2.1×10(-7); odds ratio(recessive), 2.34; P(women) = .47). The sex-specific association of rs6742078 was confirmed in samples from South America (P(men) = .046; odds ratio(recessive), 2.19; P(women) = .96).
The UGT1A1 Gilbert syndrome variant rs6742078 is associated with gallstone disease in men; further studies are required regarding the sex-specific physiology of bilirubin and bile acid metabolism. Variants of ABCG8 and UGT1A1 are the 2 major risk factors for overall gallstone disease, they contribute a population attributable risk of 21.2% among men.
Gastroenterology 12/2010; 139(6):1942-1951.e2. · 11.68 Impact Factor
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ABSTRACT: Caspase activating and recruitment domain 8 (CARD8) has been implicated as a co-regulator of several pro-inflammatory and apoptotic signaling pathways. In the present study, we demonstrate a specific modulation of NOD2-induced signaling by CARD8 in intestinal epithelial cells. We show that CARD8 physically interacts with NOD2 and inhibits nodosome assembly and subsequent signaling upon muramyl-dipeptide stimulation. Furthermore, CARD8 inhibits the direct bactericidal effect of NOD2 against intracellular infection by Listeria monocytogenes. Thus, CARD8 represents a novel molecular switch involved in the endogenous regulation of NOD2-dependent inflammatory processes in epithelial cells.
Journal of Biological Chemistry 04/2010; 285(26):19921-6. · 4.77 Impact Factor
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Nikolaus Möckelmann,
Witigo von Schönfels,
Stephan Buch, Oliver von Kampen,
Bence Sipos,
Jan Hendrik Egberts,
Philip Rosenstiel,
Andre Franke,
Mario Brosch,
Sebastian Hinz,
Christian Röder,
Holger Kalthoff,
Ulrich R Fölsch,
Michael Krawczak,
Stefan Schreiber,
Clemens Dieter Bröring,
Jürgen Tepel,
Clemens Schafmayer,
Jochen Hampe
[show abstract]
[hide abstract]
ABSTRACT: Variation in genes involved in the innate immune response may play a role in the predisposition to colorectal cancer (CRC). Several polymorphisms of the CARD15 gene (caspase activating recruitment domain, member 15) have been reported to be associated with an increased susceptibility to Crohn disease. Since the CARD15 gene product and other CARD proteins function in innate immunity, we investigated the impact of germline variation at the CARD4, CARD8 and CARD15 loci on the risk for sporadic CRC, using a large patient sample from Northern Germany.
A total of 1044 patients who had been operated with sporadic colorectal carcinoma (median age at diagnosis: 59 years) were recruited and compared to 724 sex-matched, population-based control individuals (median age: 68 years). Genetic investigation was carried out following both a coding SNP and haplotype tagging approach. Subgroup analyses for N = 143 patients with early manifestation of CRC (<or=50 age at diagnosis) were performed for all CARD loci and subgroup analyses for diverse age strata were carried out for CARD15 mutations R702W, G908R and L1007fs. In addition, all SNPs were tested for association with disease presentation and family history of CRC.
No significant differences were observed between the patient and control allelic or haplotypic spectra of the three genes under study for the total cohort (N = 1044 patients). None of the analysed SNPs was significantly associated with either tumour location or yielded significant association in the familial or non-familial CRC patient subgroups. However, in a patient subgroup (<or=45 age at diagnosis) with early disease manifestation the mutant allele of CARD15 R702W was found to be significantly associated with disease susceptibility (9.7% in cases vs 4.6% in controls; P(allelic) = 0.008, P(genotypic) = 0.0008, OR(allelic) = 2.22 (1.21-4.05) OR(ressessive) = 21.9 (1.96-245.4).
Variation in the innate immunity genes CARD4, CARD8 and CARD15 is unlikely to play a major role in the susceptibility to CRC in the German population. But, we report a significant disease contribution of CARD15 for CRC patients with very early disease manifestation, mainly driven by variant R702W.
BMC Gastroenterology 01/2009; 9:79. · 2.42 Impact Factor
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ABSTRACT: Transcription in Archaea is directed by a pol II-like RNA polymerase and homologues of TBP and TFIIB (TFB) but the crystal structure of the archaeal enzyme and the subunits involved in recruitment of RNA polymerase to the promoter-TBP-TFB-complex are unknown. We described here the cloning expression and purification of 11 bacterially expressed subunits of the Pyrococcus furiosus RNAP. Protein interactions of subunits with each other and of archaeal transcription factors TFB and TFB with RNAP subunits were studied by Far-Western blotting and reconstitution of subcomplexes from single subunits in solution. In silico comparison of a consensus sequence of archaeal RNAP subunits with the sequence of yeast pol II subunits revealed a high degree of conservation of domains of the enzymes forming the cleft and catalytic center of the enzyme. Interaction studies with the large subunits were complicated by the low solubility of isolated subunits B, A', and A'', but an interaction network of the smaller subunits of the enzyme was established. Far-Western analyses identified subunit D as structurally important key polypeptide of RNAP involved in interactions with subunits B, L, N, and P and revealed also a strong interaction of subunits E' and F. Stable complexes consisting of subunits E' and F, of D and L and a BDLNP-subcomplex were reconstituted and purified. Gel shift analyses revealed an association of the BDLNP subcomplex with promoter-bound TBP-TFB. These results suggest a major role of subunit B (Rpb2) in RNAP recruitment to the TBP-TFB promoter complex.
Journal of Biological Chemistry 11/2006; 281(41):30581-92. · 4.77 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Transcription in Archaea is directed by a pol II-like RNA polymerase and homologues of TBP and TFIIB (TFB) but the crystal
structure of the archaeal enzyme and the subunits involved in recruitment of RNA polymerase to the promoter-TBP-TFB-complex
are unknown. We described here the cloning expression and purification of 11 bacterially expressed subunits of the Pyrococcus furiosus RNAP. Protein interactions of subunits with each other and of archaeal transcription factors TFB and TFB with RNAP subunits
were studied by Far-Western blotting and reconstitution of subcomplexes from single subunits in solution. In silico comparison of a consensus sequence of archaeal RNAP subunits with the sequence of yeast pol II subunits revealed a high degree
of conservation of domains of the enzymes forming the cleft and catalytic center of the enzyme. Interaction studies with the
large subunits were complicated by the low solubility of isolated subunits B, A′, and A″, but an interaction network of the
smaller subunits of the enzyme was established. Far-Western analyses identified subunit D as structurally important key polypeptide
of RNAP involved in interactions with subunits B, L, N, and P and revealed also a strong interaction of subunits E′ and F.
Stable complexes consisting of subunits E′ and F, of D and L and a BDLNP-subcomplex were reconstituted and purified. Gel shift
analyses revealed an association of the BDLNP subcomplex with promoter-bound TBP-TFB. These results suggest a major role of
subunit B (Rpb2) in RNAP recruitment to the TBP-TFB promoter complex.
Journal of Biological Chemistry 10/2006; 281(41):30581-30592. · 4.77 Impact Factor
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Philip Rosenstiel,
Klaus Huse,
Andreas Till,
Jochen Hampe,
Stephan Hellmig,
Christian Sina,
Susanne Billmann, Oliver von Kampen,
Georg H Waetzig,
Matthias Platzer,
Dirk Seegert,
Stefan Schreiber
[show abstract]
[hide abstract]
ABSTRACT: Alterations in splicing patterns of genes contribute to the regulation of gene function by generating endogenous inhibitor or activator molecules. Nucleotide-binding and oligomerization domain (NOD) 2 is an intracellular receptor for bacterial cell wall components and plays an important role in initiating immune responses against cytoinvasive pathogens. NOD2 overexpression sensitizes intestinal epithelial cells toward bacterial cell wall components, activates the proinflammatory transcription factor NF-kappaB, and induces the subsequent release of the chemotactic cytokine IL-8. Here, we have assessed the regulation and function of a transcript isoform of NOD2, NOD2-S, generated by the skipping of the third exon, which encodes for a protein that is truncated within the second caspase recruitment (CARD) domain. NOD2-S is preferentially expressed in the human colon and is up-regulated by the antiinflammatory cytokine IL-10. Overexpression of NOD2-S down-regulates NOD2-induced NF-kappaB activation and IL-8 release. Moreover, NOD2-S also interferes with the maturation and secretion of pro-IL-1beta downstream of NOD2 and its adaptor molecule receptor-interacting protein kinase 2. We provide a molecular basis for these effects, as we show that NOD2-S interacts with both, NOD2 and receptor-interacting protein kinase 2 and inhibits the "nodosome" assembly by interfering with the oligomerization of NOD2. These data unveil another level of complexicity in the regulation of intracellular innate immunity and may have important implications for the molecular understanding of NOD/NALP protein-driven disease pathophysiology.
Proceedings of the National Academy of Sciences 03/2006; 103(9):3280-5. · 9.68 Impact Factor
