Jan Hendrik Egberts

DNA Methylation Analysis in Nonalcoholic Fatty Liver Disease Suggests Distinct Disease-Specific and Remodeling Signatures after Bariatric Surgery

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder in industrialized countries. Liver samples from morbidly obese patients (n = 45) with all stages of NAFLD and controls (n = 18) were analyzed by array-based DNA methylation and mRNA expression profiling. NAFLD-specific expression and methylation differences were seen for nine genes coding for key enzymes in intermediate metabolism (including PC, ACLY, and PLCG1) and insulin/insulin-like signaling (including IGF1, IGFBP2, and PRKCE) and replicated by bisulfite pyrosequening (independent n = 39). Transcription factor binding sites at NAFLD-specific CpG sites were >1,000-fold enriched for ZNF274, PGC1A, and SREBP2. Intraindividual comparison of liver biopsies before and after bariatric surgery showed NAFLD-associated methylation changes to be partially reversible. Postbariatric and NAFLD-specific methylation signatures were clearly distinct both in gene ontology and transcription factor binding site analyses, with >400-fold enrichment of NRF1, HSF1, and ESRRA sites. Our findings provide an example of treatment-induced epigenetic organ remodeling in humans.

Loci From a Genome-Wide Analysis of Bilirubin Levels Are Associated With Gallstone Risk and Composition

BACKGROUND & AIMS 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. METHODS 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. RESULTS 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). CONCLUSIONS 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.

Nuclear Death Receptor TRAIL-R2 Inhibits Maturation of Let-7 and Promotes Proliferation of Pancreatic and Other Tumor Cells

BACKGROUND & AIMS Tumor necrosis factor-related apoptosis inducing ligand (TRAIL-R1) (TNFRSF10A) and TRAIL-R2 (TNFRSF10B) on the plasma membrane bind ligands that activate apoptotic and other signaling pathways. Cancer cells also might have TRAIL-R2 in the cytoplasm or nucleus, although little is known about its activities in these locations. We investigated the functions of nuclear TRAIL-R2 in cancer cell lines. METHODS Proteins that interact with TRAIL-R2 initially were identified in pancreatic cancer cells by immunoprecipitation, mass spectrometry, and immunofluorescence analyses. Findings were validated in colon, renal, lung, and breast cancer cells. Functions of TRAIL-R2 were determined from small interfering RNA knockdown, real-time polymerase chain reaction, Drosha-activity, microRNA array, proliferation, differentiation, and immunoblot experiments. We assessed the effects of TRAIL-R2 overexpression or knockdown in human pancreatic ductal adenocarcinoma (PDAC) cells and their ability to form tumors in mice. We also analyzed levels of TRAIL-R2 in sections of PDACs and non-neoplastic peritumoral ducts from patients. RESULTS TRAIL-R2 was found to interact with the core microprocessor components Drosha and DGCR8 and the associated regulatory proteins p68, hnRNPA1, NF45, and NF90 in nuclei of PDAC and other tumor cells. Knockdown of TRAIL-R2 increased Drosha-mediated processing of the let-7 microRNA precursor primary let-7 (resulting in increased levels of mature let-7), reduced levels of the let-7 targets (LIN28B and HMGA2), and inhibited cell proliferation. PDAC tissues from patients had higher levels of nuclear TRAIL-R2 than non-neoplastic pancreatic tissue, which correlated with increased nuclear levels of HMGA2 and poor outcomes. Knockdown of TRAIL-R2 in PDAC cells slowed their growth as orthotopic tumors in mice. Reduced nuclear levels of TRAIL-R2 in cultured pancreatic epithelial cells promoted their differentiation. CONCLUSIONS Nuclear TRAIL-R2 inhibits maturation of the microRNA let-7 in pancreatic cancer cell lines and increases their proliferation. Pancreatic tumor samples have increased levels of nuclear TRAIL-R2, which correlate with poor outcome of patients. These findings indicate that in the nucleus, death receptors can function as tumor promoters and might be therapeutic targets.

Genetic investigation of DNA‐repair pathway genes PMS2, MLH1, MSH2, MSH6, MUTYH, OGG1 and MTH1 in sporadic colon cancer

Mutations in DNA repair genes have previously been identified as causative factors for hereditary nonpolyposis colon cancer (HNPCC). Recent evidence also supports an association between DNA sequence variation in these genes and sporadic colorectal carcinoma (CRC). Genetic investigation of DNA repair genes PMS2, MLH1, MSH2, MSH6, MUTYH, OGG1 and MTH1, as possible susceptibility factors for sporadic CRC, was done using both a haplotype tagging and a candidate (i.e. coding) single nucleotide polymorphism (SNP) approach. Some 1,068 patients with operated CRC (median age at diagnosis: 59 years) were compared to 738 sex‐matched control individuals (median age: 67 years). Haplotype tagging SNPs, previously reported risk variants and all known coding SNPs with a minor allele frequency >0.005 were genotyped in PMS2 (N = 10), MLH1 (N = 11), MSH2 (N = 18), MSH6 (N = 15), MUTYH (N = 7), OGG1 (N = 11) and MTH1 (N = 3). No evidence for an association between CRC and any of the 7 genes was detected, neither with the tagging or coding SNPs nor in a sliding window haplotype analysis (all nominal p‐values >0.05). The previously reported risk variants D132H in MLH1 and R154H in OGG1 were not even observed in the German population. Genetic CRC risk factors so far identified in DNA repair genes seem to be rare and population‐specific. Their association with the disease could not be replicated in German CRC samples. It remains to be elucidated by more systematic, large‐scale experiments whether common variants in the same genes, but present across populations, represent risk factors for sporadic CRC.

Genetic and functional identification of the likely causative variant for cholesterol gallstone disease at the ABCG5/8 lithogenic locus†

The sterolin locus (ABCG5/ABCG8) confers 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. Genetic mapping utilized patient samples from Germany (2,808 cases, 2,089 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 complementary DNA (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 localization of allelic constructs. Through fine mapping in German and Chilean samples, an ∼250 kB 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 single nucleotide polymorphisms. 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 pairwise linkage disequilibrium (r2 = 0.95). [3H]‐cholesterol export assays of allelic constructs harboring 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 “postgenomic” and “pregenomic” pathophysiological knowledge about this common complex disorder. (HEPATOLOGY 2012)

Investigation of the colorectal cancer susceptibility region on chromosome 8q24.21 in a large German case-control sample.

Human chromosome 8q24.21 has been implicated as a susceptibility region for colorectal cancer (CRC) as a result of genome‐wide association and candidate gene studies. To assess the impact of molecular variants at 8q24.21 upon the CRC risk of German individuals and to refine the disease‐associated region, a total of 2,713 patients with operated CRC (median age at diagnosis: 63 years) were compared with 2,718 sex‐matched control individuals (median age at inclusion: 65 years). Information on microsatellite instability in tumors was available for 901 patients. Association analysis of SNPs rs10505477 and rs6983267 yielded allelic p‐values of 1.42 × 10−7 and 2.57 × 10−7, respectively. For both polymorphisms, the odds ratio was estimated to be 1.50 (95% CI: 1.29–1.75) under a recessive disease model. The strongest candidate interval, outside of which significance dropped by more than 4 orders of magnitude, was delineated by SNPs rs10505477 and rs7014346 and comprised 17 kb. In a subgroup analysis, the disease association was found to be more pronounced in MSI‐stable tumors (odds ratio: 1.71). Our study confirms the role of genetic variation at 8q24.21 as a risk factor for CRC and localizes the corresponding susceptibility gene to a 17 kb candidate region.

Investigation of innate immunity genes CARD4, CARD8 and CARD15 as germline susceptibility factors for colorectal cancer.

BackgroundVariation 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.MethodsA 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 (≤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.ResultsNo 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 (≤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; Pallelic = 0.008, Pgenotypic = 0.0008, ORallelic = 2.22 (1.21-4.05) ORressessive = 21.9 (1.96-245.4).ConclusionVariation 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.

Epithelial calcineurin controls microbiota-dependent intestinal tumor development

Inflammation-associated pathways are active in intestinal epithelial cells (IECs) and contribute to the pathogenesis of colorectal cancer (CRC). Calcineurin, a phosphatase required for the activation of the nuclear factor of activated T cells (NFAT) family of transcription factors, shows increased expression in CRC. We therefore investigated the role of calcineurin in intestinal tumor development. We demonstrate that calcineurin and NFAT factors are constitutively expressed by primary IECs and selectively activated in intestinal tumors as a result of impaired stratification of the tumor-associated microbiota and toll-like receptor signaling. Epithelial calcineurin supports the survival and proliferation of cancer stem cells in an NFAT-dependent manner and promotes the development of intestinal tumors in mice. Moreover, somatic mutations that have been identified in human CRC are associated with constitutive activation of calcineurin, whereas nuclear translocation of NFAT is associated with increased death from CRC. These findings highlight an epithelial cell–intrinsic pathway that integrates signals derived from the commensal microbiota to promote intestinal tumor development.

Lack of CCR7 expression is rate limiting for lymphatic spread of pancreatic ductal adenocarcinoma

CCR7 expression on tumor cells promotes lymphatic spread in several malignant tumors. However, a comprehensive characterization of the CCL19/CCL21–CCR7 axis in pancreatic ductal adenocarcinoma (PDAC), which is known for its high rates of lymph‐node metastases, is still lacking. CCR7 mRNA and CCR7 protein were found to be expressed in spheroid cultures of all six examined PDAC cell lines. In migration assays, CCR7 expressing PDAC cells showed enhanced migration toward CCL19 and CCL21, the two ligands of CCR7. In an orthotopic nude mouse model, CCR7‐transfected PT45P1 cells gave rise to significantly larger tumors and showed a higher frequency of lymph vessel invasion and lymph‐node metastases than mock‐transfected cells. In an analysis using quantitative real‐time PCR, CCR7 showed fourfold overexpression in microdissected PDAC cells compared to normal duct cells. Moderate‐to‐strong immunohistochemical CCR7 expression, found in 58 of 121 well‐characterized human PDACs, correlated with high rates of lymph vessel invasion. Conversely, PDACs completely lacking CCR7 expression showed only low rates of lymph vessel invasion and lymph‐node metastases. The evaluation of CCL21 expression by immunofluorescence staining revealed a significant upregulation of CCL21 in peritumoral and intratumoral lymph vessels compared to lymph vessels in disease‐free pancreata. In conclusion, our study revealed strong evidence that lack of CCR7 impairs the metastatic potential of PDAC. Lymph vessel invasion by CCR7 expressing PDAC cells may be additionally enhanced by upregulation of CCL21 in tumor‐associated lymph vessels, representing a previously unknown factor of lymphatic spread.

Polymorphisms in the mitochondrial oxidative phosphorylation chain genes as prognostic markers for colorectal cancer

BackgroundCurrently, the TNM classification of malignant tumours based on clinicopathological staging remains the standard for colorectal cancer (CRC) prognostication. Recently, we identified the mitochondrial oxidative phosphorylation chain as a consistently overrepresented category in the published gene expression profiling (GEP) studies on CRC prognosis.MethodsWe evaluated associations of putative regulatory single nucleotide polymorphisms (SNPs) in genes from the oxidative phosphorylation chain with survival and disease prognosis in 613 CRC patients from Northern Germany (PopGen cohort).ResultsTwo SNPs in the 3′ untranslated region of UQCRB (complex III), rs7836698 and rs10504961, were associated with overall survival (HR = 0.52, 95% CI 0.32–0.85 and HR = 0.64, 95% CI 0.42–0.99, for TT carriers). These associations were restricted to the group of patients with cancer located in the colon (HR = 0.42, 95% CI 0.22–0.82 and HR = 0.46, 95% CI 0.25–0.83). Multivariate analysis indicated that both markers might act as independent prognostic markers. Additionally, the TT carriers were ~2 times more likely to develop tumours in the colon than in the rectum. Two SNPs in COX6B1 (complex IV) were associated with lymph node metastasis in a dominant model (rs6510502, OR = 1.75, 95% CI 1.20–2.57; rs10420252, OR = 1.68, 95% CI 1.11–2.53); rs6510502 was associated also with distant metastasis (OR = 1.67, 95% CI 1.09–2.56 in a dominant model).ConclusionsThis is the first report suggesting that markers in genes from the mitochondrial oxidative chain might be prognostic factors for CRC. Additional studies replicating the presented findings are needed.