Andreas Teufel

Genome-Wide Association Analysis in Primary Sclerosing Cholangitis

BACKGROUND & AIMS We aimed to characterize the genetic susceptibility to primary sclerosing cholangitis (PSC) by means of a genome-wide association analysis of single nucleotide polymorphism (SNP) markers. METHODS A total of 443,816 SNPs on the Affymetrix SNP Array 5.0 (Affymetrix, Santa Clara, CA) were genotyped in 285 Norwegian PSC patients and 298 healthy controls. Associations detected in this discovery panel were re-examined in independent case-control panels from Scandinavia (137 PSC cases and 368 controls), Belgium/The Netherlands (229 PSC cases and 735 controls), and Germany (400 cases and 1832 controls). RESULTS The strongest associations were detected near HLA-B at chromosome 6p21 (rs3099844: odds ratio [OR], 4.8; 95% confidence interval [CI], 3.6-6.5; P = 2.6 x 10(-26); and rs2844559: OR, 4.7; 95% CI, 3.5-6.4; P = 4.2 x 10(-26) in the discovery panel). Outside the HLA complex, rs9524260 at chromosome 13q31 showed significant associations in 3 of 4 study panels. Lentiviral silencing of glypican 6, encoded at this locus, led to the up-regulation of proinflammatory markers in a cholangiocyte cell line. Of 15 established ulcerative colitis susceptibility loci, significant replication was obtained at chromosomes 2q35 and 3p21 (rs12612347: OR, 1.26; 95% CI, 1.06-1.50; and rs3197999: OR, 1.22; 95% CI, 1.02-1.47, respectively), with circumstantial evidence supporting the G-protein-coupled bile acid receptor 1 and macrophage-stimulating 1, respectively, as the likely disease genes. CONCLUSIONS Strong HLA associations and a subset of genes involved in bile homeostasis and other inflammatory conditions constitute key components of the genetic architecture of PSC.

Genome-wide association analysis in primary sclerosing cholangitis identifies two non-HLA susceptibility loci.

Primary sclerosing cholangitis (PSC) is a chronic bile duct disease affecting 2.4–7.5% of individuals with inflammatory bowel disease. We performed a genome-wide association analysis of 2,466,182 SNPs in 715 individuals with PSC and 2,962 controls, followed by replication in 1,025 PSC cases and 2,174 controls. We detected non-HLA associations at rs3197999 in MST1 and rs6720394 near BCL2L11 (combined P = 1.1 × 10−16 and P = 4.1 × 10−8, respectively).

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.

Safety and efficacy of sorafenib in patients with advanced hepatocellular carcinoma in consideration of concomitant stage of liver cirrhosis.

Goals and Background The multikinase inhibitor sorafenib provides survival benefit for patients with advanced hepatocellular carcinoma (HCC) and liver cirrhosis (LCI) Child-Pugh A. We report our experiences with sorafenib in advanced HCC, particularly in patients with LCI Child-Pugh B/C, where only limited data are available in regard to safety and efficacy of sorafenib. Methods Thirty-four patients with advanced HCC were treated with sorafenib regardless of liver function and prior anticancer therapy. Adverse events (AEs) were graded using Common Toxicity Criteria version 3.0, tumor response was assessed according to Response Evaluation Criteria in Solid Tumors. Results Fifteen patients presented without LCI or with LCI Child-Pugh A, 15/4 patients had LCI Child-Pugh B/C. Barcelona Clinic Liver Cancer stage was B/C/D in 4/22/8 patients. During treatment period (median 2.2 mo), therapy was discontinued in 61.8% of patients due to tumor progression (32.3%), death (17.6%), AEs (8.8%), or noncompliance (2.9%). Most common grade 3/4 AEs included liver dysfunction (23.5%), diarrhea (14.7%), increased lipase (8.8%), fatigue (8.8%), and hand-foot skin reaction (5.9%). Worsening liver dysfunction/failure was more frequent (P=0.036) in patients with LCI Child-Pugh B/C compared with patients with maintained liver function (no LCI/LCI Child-Pugh A). Median overall survival was 7.2 months for patients with maintained liver function versus 3.3/3.4 months for patients with LCI Child-Pugh B/C. Conclusions These data do not support the use of sorafenib in patients with LCI Child-Pugh C, and patients with LCI Child-Pugh B should be treated with caution until larger trials provide more safety data and a clinically relevant survival benefit under sorafenib therapy.

Extended analysis of a genome-wide association study in primary sclerosing cholangitis detects multiple novel risk loci

BACKGROUND & AIMS A limited number of genetic risk factors have been reported in primary sclerosing cholangitis (PSC). To discover further genetic susceptibility factors for PSC, we followed up on a second tier of single nucleotide polymorphisms (SNPs) from a genome-wide association study (GWAS). METHODS We analyzed 45 SNPs in 1221 PSC cases and 3508 controls. The association results from the replication analysis and the original GWAS (715 PSC cases and 2962 controls) were combined in a meta-analysis comprising 1936 PSC cases and 6470 controls. We performed an analysis of bile microbial community composition in 39 PSC patients by 16S rRNA sequencing. RESULTS Seventeen SNPs representing 12 distinct genetic loci achieved nominal significance (p(replication) <0.05) in the replication. The most robust novel association was detected at chromosome 1p36 (rs3748816; p(combined)=2.1 × 10(-8)) where the MMEL1 and TNFRSF14 genes represent potential disease genes. Eight additional novel loci showed suggestive evidence of association (p(repl) <0.05). FUT2 at chromosome 19q13 (rs602662; p(comb)=1.9 × 10(-6), rs281377; p(comb)=2.1 × 10(-6) and rs601338; p(comb)=2.7 × 10(-6)) is notable due to its implication in altered susceptibility to infectious agents. We found that FUT2 secretor status and genotype defined by rs601338 significantly influence biliary microbial community composition in PSC patients. CONCLUSIONS We identify multiple new PSC risk loci by extended analysis of a PSC GWAS. FUT2 genotype needs to be taken into account when assessing the influence of microbiota on biliary pathology in PSC.

RNA-Seq Atlas – A reference database for gene expression profiling in normal tissue by next generation sequencing

MOTIVATION Next-generation sequencing technology enables an entirely new perspective for clinical research and will speed up personalized medicine. In contrast to microarray-based approaches, RNA-Seq analysis provides a much more comprehensive and unbiased view of gene expression. Although the perspective is clear and the long-term success of this new technology obvious, bioinformatics resources making these data easily available especially to the biomedical research community are still evolving. RESULTS We have generated RNA-Seq Atlas, a web-based repository of RNA-Seq gene expression profiles and query tools. The website offers open and easy access to RNA-Seq gene expression profiles and tools to both compare tissues and find genes with specific expression patterns. To enlarge the scope of the RNA-Seq Atlas, the data were linked to common functional and genetic databases, in particular offering information on the respective gene, signaling pathway analysis and evaluation of biological functions by means of gene ontologies. Additionally, data were linked to several microarray gene profiles, including BioGPS normal tissue profiles and NCI60 cancer cell line expression data. Our data search interface allows an integrative detailed comparison between our RNA-Seq data and the microarray information. This is the first database providing data mining tools and open access to large scale RNA-Seq expression profiles. Its applications will be versatile, as it will be beneficial in identifying tissue specific genes and expression profiles, comparison of gene expression profiles among diverse tissues, but also systems biology approaches linking tissue function to gene expression changes. AVAILABILITY AND IMPLEMENTATION http://medicalgenomics.org/rna_seq_atlas.

Functional ablation of the mouse Ldb1 gene results in severe patterning defects during gastrulation

The LIM domain-binding protein 1 (Ldb1) is found in multi-protein complexes containing various combinations of LIM-homeodomain, LIM-only, bHLH, GATA and Otx transcription factors. These proteins exert key functions during embryogenesis. Here we show that targeted deletion of the Ldb1 gene in mice results in a pleiotropic phenotype. There is no heart anlage and head structures are truncated anterior to the hindbrain. In about 40% of the mutants, posterior axis duplication is observed. There are also severe defects in mesoderm-derived extraembryonic structures, including the allantois, blood islands of the yolk sack, primordial germ cells and the amnion. Abnormal organizer gene expression during gastrulation may account for the observed axis defects in Ldb1 mutant embryos. The expression of several Wnt inhibitors is curtailed in the mutant, suggesting that Wnt pathways may be involved in axial patterning regulated by Ldb1.

Concurrent autoimmune diseases in patients with autoimmune hepatitis.

Background Although the pathomechanisms of autoimmune diseases in various organs remain unresolved, an accumulation of autoimmune diseases in individual patients has been observed. An overlap of autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC) or primary sclerosing cirrhosis has been well documented. However, the overlap of autoimmune diseases other than PBC or PSC has not yet been investigated in a large cohort. Goal The goal of our analysis was to investigate the incidence of concurrent autoimmune diseases in patients with AIH. Study We analyzed our cohort of 278 patients with AIH for concurrent autoimmune diseases. Results A total of 111 patients (40%) were diagnosed with additional autoimmune diseases. Besides overlap syndromes for PBC and PSC, autoimmune thyroiditis was the most common concurrent disease (28 patients, 10%). Other concurrent autoimmune diseases comprised vitiligo (5 patients), rheumatoid arthritis (5 patients), Sjogren syndrome (4 patients), ulcerative colitis (4 patients), conjunctivitis (4 patients), celiac disease (3 patients), systemic lupus erythematodes (2 patients), type I diabetes (2 patients), multiple sclerosis (2 patients), polymyalgia rheumatica (2 patients), and urticaria (2 patients). One patient each was diagnosed with Crohns disease, autoimmune gastritis, collagenous colitis, hypophysitis, and sarcoidosis. Investigating 100 patients with polyglandular syndrome and autoimmune thyroid disease for the occurrence of autoantibodies associated with AIH, we identified AIH-associated antibodies only in 1 patient. Conclusions Concurrent autoimmune diseases are common in patients with AIH and mirror the full range of known autoimmune diseases. Therefore, an extended diagnostic screening for accumulating autoimmune diseases, especially autoimmune thyroiditis, seems reasonable in patients with AIH.

Knockout of myeloid cell leukemia‐1 induces liver damage and increases apoptosis susceptibility of murine hepatocytes

Myeloid cell leukemia‐1 (Mcl‐1) is an antiapoptotic member of the Bcl‐2 protein family. It interacts with proapoptotic Bcl‐2 family members, thereby inhibiting mitochondrial activation and induction of apoptosis. Mcl‐1 is essential for embryonal development and the maintenance of B cells, T cells, and hematopoietic stem cells. We have recently shown that induction of Mcl‐1 by growth factors rescues primary human hepatocytes from CD95‐mediated apoptosis. This prompted us to further analyze the relevance of Mcl‐1 for hepatocellular homeostasis. Therefore, we generated a hepatocyte‐specific Mcl‐1 knockout mouse (Mcl‐1flox/flox‐AlbCre). Deletion of Mcl‐1 in hepatocytes results in liver cell damage caused by spontaneous induction of apoptosis. Livers of Mcl‐1flox/flox‐AlbCre mice are smaller compared to control littermates, due to higher apoptosis rates. As a compensatory mechanism, proliferation of hepatocytes is enhanced in the absence of Mcl‐1. Importantly, hepatic pericellular fibrosis occurs in Mcl‐1 negative livers in response to chronic liver damage. Furthermore, Mcl‐1flox/flox‐AlbCre mice are more susceptible to hepatocellular damage induced by agonistic anti‐CD95 antibodies or concanavalin A. Conclusion: The present study provides in vivo evidence that Mcl‐1 is a crucial antiapoptotic factor for the liver, contributing to hepatocellular homeostasis and protecting hepatocytes from apoptosis induction. (HEPATOLOGY 2009.)

Genome-Wide Association Analysis in Primary Sclerosing Cholangitis and Ulcerative Colitis Identifies Risk Loci at GPR35 and TCF4

Approximately 60%‐80% of patients with primary sclerosing cholangitis (PSC) have concurrent ulcerative colitis (UC). Previous genome‐wide association studies (GWAS) in PSC have detected a number of susceptibility loci that also show associations in UC and other immune‐mediated diseases. We aimed to systematically compare genetic associations in PSC with genotype data in UC patients with the aim of detecting new susceptibility loci for PSC. We performed combined analyses of GWAS for PSC and UC comprising 392 PSC cases, 987 UC cases, and 2,977 controls and followed up top association signals in an additional 1,012 PSC cases, 4,444 UC cases, and 11,659 controls. We discovered novel genome‐wide significant associations with PSC at 2q37 [rs3749171 at G‐protein‐coupled receptor 35 (GPR35); P = 3.0 × 10−9 in the overall study population, combined odds ratio [OR] and 95% confidence interval [CI] of 1.39 (1.24‐1.55)] and at 18q21 [rs1452787 at transcription factor 4 (TCF4); P = 2.61 × 10−8, OR (95% CI) = 0.75 (0.68‐0.83)]. In addition, several suggestive PSC associations were detected. The GPR35 rs3749171 is a missense single nucleotide polymorphism resulting in a shift from threonine to methionine. Structural modeling showed that rs3749171 is located in the third transmembrane helix of GPR35 and could possibly alter efficiency of signaling through the GPR35 receptor. Conclusion: By refining the analysis of a PSC GWAS by parallel assessments in a UC GWAS, we were able to detect two novel risk loci at genome‐wide significance levels. GPR35 shows associations in both UC and PSC, whereas TCF4 represents a PSC risk locus not associated with UC. Both loci may represent previously unexplored aspects of PSC pathogenesis. (HEPATOLOGY 2013;58:1074–1083)