Crohn’s disease (CD), a form of inflammatory bowel disease, has a higher prevalence in Ashkenazi Jewish than in non-Jewish European populations. To define the role of non-synonymous mutations, we performed exome sequencing of Ashkenazi Jewish patients with CD, followed by array-based genotyping and association analysis in 2,066 CD cases and 3,633 healthy controls. We detected association signals in the LRRK2 gene that conferred CD risk (N2081D variant, P=9.5×10−10) or protection (N551K variant, tagging R1398H-associated haplotype, P=3.3×10−8). These variants affected CD age of onset, disease location, LRRK2 activity, and autophagy. Bayesian network analysis of CD patient intestinal tissue further implicated LRRK2 in CD pathogenesis. Analysis of the extended LRRK2 locus in 24,570 CD cases, patients with Parkinson’s disease (PD), and healthy controls revealed extensive pleiotropy, with similar genetic effects between CD and PD in both Ashkenazi Jewish and non-Jewish cohorts. The LRRK2 N2081D CD risk allele is located in the same kinase domain as G2019S, a mutation that is the major genetic cause of familial and sporadic PD. Like the G2019S mutation, the N2081D variant is associated with increased kinase activity, whereas neither N551K nor R1398H on the protective haplotype altered kinase activity. R1398H, but not N551K, increased GTPase activity, thereby deactivating LRRK2. The presence of shared LRRK2 alleles in CD and PD provides refined insight into disease mechanisms and may have major implications for the treatment of these two seemingly unrelated diseases.

Transforming growth factor (TGF)-b1 has a biphasic effect on rat intestinal epithelial (RIE) cells. By itself, TGF-b1 functions as a tumor suppressor by inhibiting the growth, migration and invasion of RIE cells. We show in this study that in conjunction with epidermal growth factor (EGF), TGF-b1 helped to augment migration, invasion and anchorage-independent growth (AIG) compared to that by EGF alone. EGF plus TGF-b1 induced a dramatic morphological change characteristic of epithelial-mesenchymal transition (EMT). The mechanism for this enhanced effect of TGF-b1 and EGF on oncogenic properties was explored by analysis of EGF-and TGF-b1-mediated signaling pathways and complementary DNA arrays. TGF-b1 augmented EGF-mediated signaling of mitogen-activated protein kinase (MAPK) and AKT by enhancing and prolonging the activation of the former and prolonging the activation of the latter. Inhibition of MAPK, but not phosphoinositide-3 kinase (PI3K), abolished TGF-b1 plus EGF-induced EMT and downregulation of E-cadherin at mRNA and protein levels. By contrast, cell migration and invasion were sensitive to inhibition of either MAPK or PI3 kinase. TGF-b1 plus EGF-induced AIG was significantly more resistant to inhibition of PI3K and MAPK compared to that induced by EGF alone. EGF and TGF-b1 synergistically induced the expression of a series of proteases including matrix metalloproteinase (MMP) 1 (collagenase), MMP3, MMP9, MMP10, MMP14 and cathepsin. Among them, the expression of MMP1, MMP3, MMP9 and MMP10 was MAPK dependent. Inhibition of the MMPs or cathepsin significantly blocked EGF plus TGF-b1-induced invasion, but had no effect on colony formation.Phospholipase C (PLC) and Cox2 induced by EGF plus TGF-b1 also played a significant role in invasion, whereas PLC was also important for colony formation. Our study reveals specific signaling functions and induction of genes differentially required for enhanced effect of EGF-and TGF-b1-induced oncogenic properties, and helps to explain the tumor-promoting effect of TGF-b1 in human cancer with elevated expression or activation of TGF-b1 and receptor protein tyrosine kinases.

Background Little is known about the role of the microbiome in primary sclerosing cholangitis. Aim Our goal was to explore the mucosa-associated microbiota in PSC patients across different locations in the gut, and to compare it with IBD-only patients and healthy controls. Methods Biopsies from the terminal ileum, right colon, and left colon were collected from patients and healthy controls undergoing colonoscopy. Microbiota profiling using bacterial 16S rRNA sequencing was performed on all biopsies. Results Forty-four patients were recruited: 20 with PSC (19 with PSC-IBD and one with PSC-only), 15 with IBD-only, and 9 healthy controls. The overall microbiome profile was similar throughout different locations in the gut. No differences in the global microbiome profile were found. However, we observed significant PSC-associated enrichment in Barnesiellaceae at the family level, and in Blautia and an unidentified Barnesiellaceae at the genus level. At the operational taxa unit (OTU) level, most shifts in PSC were observed in Clostridiales and Bacteroidales orders, with approximately 86% of shifts occurring within the former order. Conclusion The overall microbiota profile was similar across multiple locations in the gut from the same individual regardless of disease status. In this study, the mucosa associated-microbiota of PSC patients was characterized by enrichment of Blautia and Barnesiellaceae and by major shifts in OTUs within Clostridiales order.

Background: Patients with primary sclerosing cholangitis associated with inflammatory bowel disease (PSC-IBD) have a very high risk of developing colorectal neoplasia. Alterations in the gut microbiota and/or gut bile acids could account for the increase in this risk. However, no studies have yet investigated the net result of cholestasis and a potentially altered bile acid pool interacting with a dysbiotic gut flora in the inflamed colon of PSC-IBD. Aim: The aim of this study was to compare the gut microbiota and stool bile acid profiles, as well as and their correlation in patients with PSC-IBD and inflammatory bowel disease alone. Methods: Thirty patients with extensive colitis (15 with concomitant primary sclerosing cholangitis) were prospectively recruited and fresh stool samples were collected. The microbiota composition in stool was profiled using bacterial 16S rRNA sequencing. Stool bile acids were assessed by high-performance liquid chromatography tandem mass spectrometry. Results: The total stool bile acid pool was significantly reduced in PSC-IBD. Although no major differences were observed in the individual bile acid species in stool, their overall combination allowed a good separation between PSC-IBD and inflammatory bowel disease. Compared with inflammatory bowel disease alone, PSC-IBD patients demonstrated a different gut microbiota composition with enrichment in Ruminococcus and Fusobacterium genus compared with inflammatory bowel disease. At the operational taxonomic unit level major shifts were observed within the Firmicutes (73%) and Bacteroidetes phyla (17%). Specific microbiota-bile acid correlations were observed in PSC-IBD, where 12% of the operational taxonomic units strongly correlated with stool bile acids, compared with only 0.4% in non-PSC-IBD. Conclusions: Patients with PSC-IBD had distinct microbiota and microbiota-stool bile acid correlations as compared with inflammatory bowel disease. Whether these changes are associated with, or may predispose to, an increased risk of colorectal neoplasia needs to be further clarified.

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