Thiopurine therapy, commonly used in autoimmune conditions, can be complicated by life-threatening leukopenia. This leukopenia is associated with genetic variation in TPMT (encoding thiopurine ...S-methyltransferase). Despite a lower frequency of TPMT mutations in Asians, the incidence of thiopurine-induced leukopenia is higher in Asians than in individuals of European descent. Here we performed an Immunochip-based 2-stage association study in 978 Korean subjects with Crohn's disease treated with thiopurines. We identified a nonsynonymous SNP in NUDT15 (encoding p.Arg139Cys) that was strongly associated with thiopurine-induced early leukopenia (odds ratio (OR) = 35.6; P(combined) = 4.88 × 10(-94)). In Koreans, this variant demonstrated sensitivity and specificity of 89.4% and 93.2%, respectively, for thiopurine-induced early leukopenia (in comparison to 12.1% and 97.6% for TPMT variants). Although rare, this SNP was also strongly associated with thiopurine-induced leukopenia in subjects with inflammatory bowel disease of European descent (OR = 9.50; P = 4.64 × 10(-4)). Thus, NUDT15 is a pharmacogenetic determinant for thiopurine-induced leukopenia in diverse populations.
Fucosyltransferase 2 (FUT2) is an enzyme that is responsible for the synthesis of the H antigen in body fluids and on the intestinal mucosa. The H antigen is an oligosaccharide moiety that acts as ...both an attachment site and carbon source for intestinal bacteria. Non-secretors, who are homozygous for the loss-of-function alleles of FUT2 gene (sese), have increased susceptibility to Crohn's disease (CD). To characterize the effect of FUT2 polymorphism on the mucosal ecosystem, we profiled the microbiome, meta-proteome and meta-metabolome of 75 endoscopic lavage samples from the cecum and sigmoid of 39 healthy subjects (12 SeSe, 18 Sese and 9 sese). Imputed metagenomic analysis revealed perturbations of energy metabolism in the microbiome of non-secretor and heterozygote individuals, notably the enrichment of carbohydrate and lipid metabolism, cofactor and vitamin metabolism and glycan biosynthesis and metabolism-related pathways, and the depletion of amino-acid biosynthesis and metabolism. Similar changes were observed in mice bearing the FUT2(-/-) genotype. Metabolomic analysis of human specimens revealed concordant as well as novel changes in the levels of several metabolites. Human metaproteomic analysis indicated that these functional changes were accompanied by sub-clinical levels of inflammation in the local intestinal mucosa. Therefore, the colonic microbiota of non-secretors is altered at both the compositional and functional levels, affecting the host mucosal state and potentially explaining the association of FUT2 genotype and CD susceptibility.
Cucurbitacins are a group of diverse triterpenoid substances isolated from plants with medicinal properties. One particularly potent family member is cucurbitacin B (CuB). The antiproliferative ...effects of CuB against human breast cancer cells were tested. Six human breast cancer cell lines were examined because they represent a diverse mix of breast cancer subtypes varying in expression of estrogen receptor (ER), Her2/neu, and p53 mutation. The antiproliferative effect of CuB were also studied in vivo. The effective dose inhibiting 50% growth (ED50) was between 10−8 M and 10−7 M for this collection of breast cancer cell lines. These cells underwent rapid morphologic changes after 15–20 min exposure to CuB (5 × 10−7 M), which was associated with disruption of the microtubules and F‐actin, as observed by confocal microscopy. Human MDA‐MB‐231 (ER‐, p53 mutated) breast cancer cells were orthotopically implanted into the breasts of nude mice who intraperitoneally received either CuB 1.0 mg/kg or vehicle. Tumor volume was reduced by 55% in the group that received CuB for 6 weeks compared with vehicle controls. No apparent organ tissue damage was observed by pathological assessment. Interestingly, the experimental mice had lower serum glucose levels, consistent with use of CuB as an antidiabetic drug in China. This drug appears to be a third in a family of drugs targeting the microtubules (taxanes e.g. taxol, vinca alkaloid e.g. vincristine, and now CuB). Our in vitro and in vivo results suggest that CuB may be an effective, new approach for the treatment of ER‐, Her2/neu amplified, and p53 mutant breast cancers. (Cancer Sci 2008; 99: 1793–1797)
Stunting is known to be heavily influenced by environmental factors, so the genetic contribution has received little attention. Here we report an exploration of genetic influences in stunted Zambian ...children with environmental enteropathy. Children with stunting (LAZ -1) from the same community as controls. Blood and stool samples were taken to measure biomarkers of intestinal inflammation, epithelial damage, and microbial translocation. Single nucleotide polymorphism array genotyping was carried out on saliva samples using the H3Africa consortium array. Genome wide associations were analysed in 117 cases and 41 controls. While no significant associations with stunting were observed at P<5x10.sup.-8, likely due to the small sample size, interesting associations were observed at lower thresholds. SNPs associated with stunting were in genomic regions known to modulate neuronal differentiation and fatty acid biosynthesis. SNPs associated with increased microbial translocation were associated with non-integrin membrane ECM interactions, tight junctions, hemostasis, and G-alpha signalling events. SNPs associated with increased inflammation were associated with, ECM interactions, purine metabolism, axon guidance, and cell motility. SNPs negatively associated with inflammation overlapped genes involved in semaphoring interactions. We explored the existing coeliac disease risk HLA genotypes and found present: DQ2.5 (7.5%), DQ8 (3.5%) and DQ2.2 (3.8%); however, no children were positive for coeliac antibodies. We detected HLA-DRB:1301 and HLA-C:1802 with high odds ratios and P<0.05 in stunted children compared to controls. Genetic variations associated with stunting and the enteropathy underlying it, include variants associated with multiple pathways relating to gene expression, glycosylation, nerve signalling, and sensing of the nutritional and microbiological milieu.
New Findings
What is the central question of this study?
Non‐responsive stunting is characterised by a progressive decline of circulating glucagon‐like peptide 2: what are the possible causes of this ...decline?
What is the main finding and its importance?
In contrast with the established loss of Paneth and goblet cells in environmental enteropathy, there was no evidence of a parallel loss of enteroendocrine cells as seen by positive tissue staining for chromogranin A. Transcriptomic and genomic analyses showed evidence of genetic transcripts that could account for some of the variability seen in circulating glucagon‐like peptide 2 values.
Nutrient sensing determines digestive and hormonal responses following nutrient ingestion. We have previously reported decreased levels of glucagon‐like peptide 2 (GLP‐2) in children with stunting. Here we demonstrate the presence of enteroendocrine cells in stunted children and explore potential pathways that may be involved in reduced circulating levels of GLP‐2. At the time of performing diagnostic endoscopies for non‐responsive stunted children, intestinal biopsies were collected for immunofluorescence staining of enteroendocrine cells and transcriptomic analysis. Circulating levels of GLP‐2 were also measured and correlated with transcriptomic data. An exploratory genome‐wide association study (GWAS) was conducted on DNA samples (n = 158) to assess genetic contribution to GLP‐2 variability. Intestinal tissue sections collected from non‐responsive stunted children stained positive for chromogranin A (88/89), alongside G‐protein‐coupled receptors G‐protein receptor 119 (75/87), free fatty acid receptor 3 (76/89) and taste 1 receptor 1 (39/45). Transcriptomic analysis found three pathways correlated with circulating GLP‐2: sugar metabolism, epithelial transport, and barrier function, which likely reflect downstream events following receptor–ligand interaction. GWAS analysis revealed potential genetic contributions to GLP‐2 half‐life and receptor binding. Enteroendocrine cell loss was not identified in stunted Zambian children as has been observed for goblet and Paneth cells. Transcriptomic analysis suggests that GLP‐2 has pleiotrophic actions on the intestinal mucosa in malnutrition, but further work is needed to dissect pathways leading to perturbations in nutrient sensing.
Mechanism identified by which TL1A differentially regulates the expression of TH17 effector cytokines IL‐17 and IL‐22, in human TH17 cells.
TL1A contributes to the pathogenesis of several chronic ...inflammatory diseases, including those of the bowel by enhancing TH1, TH17, and TH2 responses. TL1A mediates a strong costimulation of these TH subsets, particularly of mucosal CCR9+ T cells. However, the signaling pathways that TL1A induces in different TH subsets are incompletely understood. We investigated the function of TL1A on human TH17 cells. TL1A, together with TGF‐β, IL‐6, and IL‐23, enhanced the secretion of IL‐17 and IFN‐γ from human CD4+ memory T cells. TL1A induced expression of the transcription factors BATF and T‐bet that correlated with the secretion of IL‐17 and IFN‐γ. In contrast, TL1A alone induced high levels of IL‐22 in memory CD4+ T cells and committed TH17 cells. However, TL1A did not enhance expression of IL‐17A in TH17 cells. Expression of the transcription factor aryl hydrocarbon receptor, which regulates the expression of IL‐22 was not affected by TL1A. Transcriptome analysis of TH17 cells revealed increased expression of IL‐9 in response to TL1A. Blocking IL‐9 receptor antibodies abrogated TL1A‐induced IL‐22 secretion. Furthermore, TL1A increased IL‐9 production by peripheral TH17 cells isolated from patients with Crohn’s disease. These data suggest that TL1A differentially induces expression of TH17 effector cytokines IL‐17, ‐9, and ‐22 and provides a potential target for therapeutic intervention in TH17‐driven chronic inflammatory diseases.
Crohn's disease (CD) patients demonstrate distinct intestinal microbial compositions and metabolic characteristics compared to unaffected controls. However, the impact of inflammation and underlying ...genetic risk on these microbial profiles and their relationship to disease phenotype are unclear. We used lavage sampling to characterize the colonic mucosal-luminal interface (MLI) microbiome of CD patients in endoscopic remission and unaffected controls relative to obesity, disease genetics, and phenotype.
Cecum and sigmoid colon were sampled from 110 non-CD controls undergoing screening colonoscopy who were stratified by body mass index and 88 CD patients in endoscopic remission (396 total samples). CD polygenic risk score (GRS) was calculated using 186 known CD variants. MLI pellets were analyzed by 16S ribosomal RNA gene sequencing, and supernatants by untargeted liquid chromatography-mass spectrometry.
CD and obesity were each associated with decreased cecal and sigmoid MLI bacterial diversity and distinct bacterial composition compared to controls, including expansion of Escherichia/Shigella. Cecal and sigmoid dysbiosis indices for CD were significantly greater in obese controls than non-overweight controls. CD, but not obesity, was characterized by altered biogeographic relationship between the sigmoid and cecum. GRS was associated with select taxonomic shifts that overlapped with changes seen in CD compared to controls including Fusobacterium enrichment. Stricturing or penetrating Crohn's disease behavior was characterized by lower MLI bacterial diversity and altered composition, including reduced Faecalibacterium, compared to uncomplicated CD. Taxonomic profiles including reduced Parasutterella were associated with clinical disease progression over a mean follow-up of 3.7 years. Random forest classifiers using MLI bacterial abundances could distinguish disease state (area under the curve (AUC) 0.93), stricturing or penetrating Crohn's disease behavior (AUC 0.82), and future clinical disease progression (AUC 0.74). CD patients showed alterations in the MLI metabolome including increased cholate:deoxycholate ratio compared to controls.
Obesity, CD in endoscopic remission, and high CD genetic risk have overlapping colonic mucosal-luminal interface (MLI) microbiome features, suggesting a shared microbiome contribution to CD and obesity which may be influenced by genetic factors. Microbial profiling during endoscopic remission predicted Crohn's disease behavior and progression, supporting that MLI sampling could offer unique insight into CD pathogenesis and provide novel prognostic biomarkers.
Background:
Acute severe ulcerative colitis (UC) remains a significant clinical challenge and the ability to predict, at an early stage, those individuals at risk of colectomy for medically ...refractory UC (MR‐UC) would be a major clinical advance. The aim of this study was to use a genome‐wide association study (GWAS) in a well‐characterized cohort of UC patients to identify genetic variation that contributes to MR‐UC.
Methods:
A GWAS comparing 324 MR‐UC patients with 537 non‐MR‐UC patients was analyzed using logistic regression and Cox proportional hazards methods. In addition, the MR‐UC patients were compared with 2601 healthy controls.
Results:
MR‐UC was associated with more extensive disease (P = 2.7 × 10−6) and a positive family history of UC (P = 0.004). A risk score based on the combination of 46 single nucleotide polymorphisms (SNPs) associated with MR‐UC explained 48% of the variance for colectomy risk in our cohort. Risk scores divided into quarters showed the risk of colectomy to be 0%, 17%, 74%, and 100% in the four groups. Comparison of the MR‐UC subjects with healthy controls confirmed the contribution of the major histocompatibility complex to severe UC (peak association: rs17207986, P = 1.4 × 10−16) and provided genome‐wide suggestive association at the TNFSF15 (TL1A) locus (peak association: rs11554257, P = 1.4 × 10−6).
Conclusions:
A SNP‐based risk scoring system, identified here by GWAS analyses, may provide a useful adjunct to clinical parameters for predicting the natural history of UC. Furthermore, discovery of genetic processes underlying disease severity may help to identify pathways for novel therapeutic intervention in severe UC. (Inflamm Bowel Dis 2010)
ZIP8 is a metal transporter with a role in manganese (Mn) homeostasis. A common genetic variant in ZIP8 (rs13107325; A391T) ranks in the top 10 of pleiotropic SNPs identified in GWAS; A391T has ...associations with an increased risk of schizophrenia, obesity, Crohn's disease, and reduced blood Mn. Here, we used CRISPR/Cas9-mediated knockin (KI) to generate a mouse model of ZIP8 A391T (Zip8 393T-KI mice). Recapitulating the SNP association with blood Mn, blood Mn was reduced in Zip8 393T-KI mice. There was restricted abnormal tissue Mn homeostasis, with decreases in liver and kidney Mn and a reciprocal increase in biliary Mn, providing in vivo evidence of hypomorphic Zip8 function. Upon challenge in a chemically induced colitis model, male Zip8 393T-KI mice exhibited enhanced disease susceptibility. ZIP8 391-Thr associated with reduced triantennary plasma N-glycan species in a population-based cohort to define a genotype-specific glycophenotype hypothesized to be linked to Mn-dependent glycosyltransferase activity. This glycophenotype was maintained in a cohort of patients with Crohn's disease. These data and the pleiotropic disease associations with ZIP8 391-Thr suggest underappreciated roles of Mn homeostasis in complex human disease.
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