Most genetic variants associated with complex diseases lie in non-coding regions1, complicating efforts to identify effector genes and relevant cell types. Here we map cis-expression quantitative trait loci (eQTLs) across 2.2 million single cells using intestinal biopsies and blood from 421 individuals, including 125 with inflammatory bowel disease (IBD). Cell-type-level eQTLs were more distal to transcription start sites, enriched in enhancers, less likely to regulate the nearest gene, and more than 3.5-fold more likely to colocalize with IBD loci detected in genome-wide association studies (GWASs) than eQTLs detected at tissue-level resolution. We nominate effector genes at more than half of known IBD loci, including MAML2, PSEN2 and ZMIZ1 in myeloid cells, implicating reduced Notch signalling in intestinal immune dysfunction. We also identify Wnt-regulated genes, including MYC, in epithelial stem and progenitor cells, suggesting that impaired renewal contributes to barrier breakdown. Our results provide a mechanistic map that links genetic risk to specific genes and cell types in IBD, and a generalized framework for interpretation of GWAS loci using single-cell eQTL mapping of disease-relevant tissues in complex diseases.

Most genetic variants associated with complex diseases lie in non-coding regions, complicating efforts to identify effector genes and relevant cell types. Here, we map cis-eQTLs across 2.2 million single cells from blood and intestinal biopsies of 421 individuals, including 125 with inflammatory bowel disease (IBD). Cell-type-level eQTLs were more distal to transcription start sites, enriched in enhancers, less likely to regulate the nearest gene, and over two-fold more likely to colocalise with IBD GWAS loci than eQTLs detected at tissue-level resolution. We nominate effector genes at over half of known IBD loci, including MAML2 , PSEN2, and ZMIZ1 in myeloid cells, implicating reduced Notch signalling in intestinal immune dysfunction. We also identify Wnt regulated genes, including MYC , in epithelial stem and progenitor cells, suggesting that impaired renewal contributes to barrier breakdown. Our results provide a mechanistic map linking genetic risk to specific genes and cell types in IBD, and a framework for effector gene discovery in complex disease.

Crohn’s disease (CD) is a complex inflammatory disorder of incompletely understood molecular aetiology. We generated a large single-cell RNA sequencing dataset from the terminal ileal biopsies of two independent cohorts comprising a total of 50 CD patients and 71 healthy controls. We performed transcriptomic analyses to reveal genes, cell types and mechanisms perturbed in CD, leveraging the power of the two cohorts to confirm our findings and assess replicability. In addition to mapping widespread alterations in cytokine signalling, we provide evidence of pan-epithelial upregulation of MHC class I genes and pathways in CD. Using non-negative matrix factorization we revealed intra- and inter-cellular upregulation of expression programs such as G-protein coupled receptor signalling and interferon signalling, respectively, in CD. We observed an enrichment of CD heritability among marker genes for various activated T cell types and myeloid cells, supporting a causal role for these cell-types in CD aetiology. Comparisons between our discovery and replication cohort revealed significant variation in differential gene-expression replicability across cell types. B, T and myeloid cells showed particularly poor replicability, suggesting caution should be exercised when interpreting unreplicated differential gene-expression result in these cell types. Overall, our results provide a rich resource for identifying cell-type specific biomarkers of Crohn’s disease and identifying genes, cell types and pathways that are causally and replicably associated with disease.

Abstract Background Under normal physiological conditions, colonic crypts accrue ~40 somatic mutations for every year of life. That somatic mutations contribute to the development of cancer is well established, but their patterns, burden and functional consequences in diseases other than cancer have not been extensively studied and our understanding of the effects of chronic inflammation on the mutational profile and clonal structure of the colon is limited. Here, we investigated how the recurrent cycles of inflammation, ulceration and regeneration seen in IBD impact the mutational and clonal structure of intestinal epithelia. Methods We isolated and whole-genome sequenced ~400 individual colonic crypts from 46 IBD patients and compared these to ~400 crypts from 41 non-IBD controls. We compared the mutation burden, mutational signature exposure, clonal structure and cancer driver mutation landscape in crypts from actively and previously inflamed regions with crypts dissected from controls. Results We estimated the base substitution rate of affected colonic epithelial cells to be doubled after IBD onset. This change was primarily driven by acceleration of mutational processes ubiquitously observed in normal colon (Figure 1), and we did not detect an IBD-specific mutational process. In contrast to the normal colon, where clonal expansions outside the confines of the crypt are rare, we observed widespread millimetre-scale clonal expansions, even in the absence of mutations in KRAS, TP53 and APC (Figure 2). We discovered that mutations in ARID1A, PIGR and ZC3H12A, and genes in the interleukin 17 and Toll-like receptor pathways, were under positive selection in colonic crypts from IBD patients (Figure 3). With the exception of ARID1A, these genes and pathways have not been previously associated with cancer risk. A previously published mouse model of ZC3H12A suggests that LoF mutations in this gene may facilitate healing of affected mucosa without promoting tumorigenesis. This could make the encoded protein an attractive drug target. The observed enrichment of mutations in PIGR and IL17 and TLR pathways suggests that somatic mutations may initiate, maintain or perpetuate IBD pathogenesis through disruption of microbe-epithelial homeostasis. Conclusion Our results provide new insights into the consequences of chronic intestinal inflammation on the mutational profile and clonal structure of colonic epithelia. We identify the mutagens driving the increase in mutation burden and mutations which are under positive selection in the context of inflammation. Our results suggest that studying somatic mutations in the colon can reveal putative drug targets and pathogenic mechanisms for IBD.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease associated with increased risk of gastrointestinal cancers. We whole-genome sequenced 446 colonic crypts from 46 IBD patients and compared these to 412 crypts from 41 non-IBD controls from our previous publication on the mutation landscape of the normal colon. The average mutation rate of affected colonic epithelial cells is 2.4-fold that of healthy colon, and this increase is mostly driven by acceleration of mutational processes ubiquitously observed in normal colon. In contrast to the normal colon, where clonal expansions outside the confines of the crypt are rare, we observed widespread millimeter-scale clonal expansions. We discovered non-synonymous mutations in ARID1A, FBXW7, PIGR, ZC3H12A, and genes in the interleukin 17 and Toll-like receptor pathways, under positive selection in IBD. These results suggest distinct selection mechanisms in the colitis-affected colon and that somatic mutations potentially play a causal role in IBD pathogenesis.

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Gastrointestinal diseases such as irritable bowel syndrome, Crohn’s disease (CD) and ulcerative colitis are a growing concern in the developed world. Current techniques for diagnosis are often costly, time consuming, inefficient, of great discomfort to the patient, and offer poor sensitivities and specificities. This paper describes the development and evaluation of a new methodology for the non-invasive diagnosis of such diseases using a combination of gas chromatography mass spectrometry (GC–MS) and chemometrics. Several potential sample matrices were tested: blood, breath, faeces and urine. Faecal samples provided the only statistically significant results, providing discrimination between CD and healthy controls with an overall classification accuracy of 85 % (78 % specificity; 93 % sensitivity). Differentiating CD from other diseases proved more challenging, with overall classification accuracy dropping to 79 % (83 % specificity; 68 % sensitivity). This diagnostic performance compares well with the gold standard technique of colonoscopy, suggesting that GC–MS may have potential as a non-invasive screening tool.

Background: The aim of this study was to determine whether volatile organic compounds (VOCs) present in the headspace of feces could be used to diagnose or distinguish between chronic diseases of the gastrointestinal tract and apparently healthy volunteers. Methods: A total of 87 people were recruited, divided between 4 categories: healthy volunteers (n = 19), Crohn’s disease (n = 22), ulcerative colitis (n = 20), and irritable bowel syndrome (n = 26). They each supplied fecal samples before, and except for the healthy volunteers, after treatment. Fecal samples were incubated in a sample bag with added purified air at 40°C and headspace samples were taken and concentrated on thermal sorption tubes. Gas chromatography–mass spectrometry then desorbed and analyzed these. The concentrations of a selection of high-abundance compounds were determined and assessed for differences in concentration between the groups. Results: Crohn's disease samples showed significant elevations in the concentrations of ester and alcohol derivates of short-chain fatty acids and indole compared with the other groups; indole and phenol were elevated in ulcerative colitis and irritable bowel syndrome but not at a statistically significant level. After treatment, the levels of many of the VOCs were significantly reduced and were more similar to those concentrations in healthy controls. Conclusions: The abundance of a number of VOCs in feces differs markedly between Crohn's disease and other gastrointestinal conditions. Following treatment, the VOC profile is altered to more closely resemble that of healthy volunteers.

The relative abundance of different groups of sulphate-reducing bacteria (SRB) in faecal DNA collected before and after therapy from patients suffering from Crohn's disease (CD), irritable bowel syndrome (IBS) or ulcerative colitis (UC) has been compared with that from healthy controls. Growth tests revealed that SRB were not more abundant in samples from patients with CD before treatment than in the healthy control group. For most of the 128 samples available, these preliminary results were confirmed using degenerate PCR primers that amplify the dsrAB gene. However, some samples from patients with CD before treatment contained a growth inhibitor that was absent from IBS or UC samples. In-depth sequencing of PCR-generated dsrB fragments revealed that the diversity detected was surprisingly low, with only eight strains of SRB and the sulphite-reducing bacterium, Bilophila wadsworthia, detected above the 0.1% threshold. The proportion of the two major species detected, B. wadsworthia and Desulfovibrio piger, was as high as 93.5% of the total SRB population in the healthy control group and lower in all patient groups. Four previously undescribed species were found: it is impossible to predict whether they are sulphate or sulphite-reducing bacteria.

School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK; Gastroenterology 6 Research Unit, Box 262, Addenbrookes Hospital, Cambridge CB2 0QQ, UK; School of Electronic, 7 Electrical and Computer Engineering, University of Birmingham, Birmingham B15 2TT, UK; 8 Cranfield Health, Vincent Building, Cranfield University, Bedfordshire MK43 0AL: The Department 9 of Life, Health and Chemical Sciences, Open University, Milton Keynes, MK7 6BJ, UK; 10

BACKGROUND:DNA methylation constitutes a key epigenetic mechanism by which cells regulate gene transcription. Among its roles are the dynamic regulation of gene expression, for example, as part of an evolving immune response, and cell differentiation in specialized tissues. Here our aim was to study the impact of differences in methylation patterns in the intestine with regard to inflammatory bowel disease (IBD) susceptibility and activity. METHODS:Having extracted DNA from rectal biopsies, we conducted genome-wide methylation profiling using the HumanMethylation27 BeadChip microarray to identify genes showing evidence of differential methylation between cases of ulcerative colitis and Crohn's disease and healthy controls. Selected methylation signals were validated in an independent replication panel by pyrosequencing. Correlation with gene expression was sought by quantitative real-time polymerase chain reaction (RT-PCR). RESULTS:Multiple genes showed significant evidence of differential methylation, several appearing in both ulcerative colitis and Crohn's disease comparisons including THRAP2, FANCC, GBGT1, DOK2, TNFSF4, TNFSF12, and FUT7. Many more than expected by chance overlapped with genes previously implicated as playing a role in IBD susceptibility in genome-wide association scans, including CARD9, ICAM3, and IL8RB (P < 0.001). Correlation between methylation and gene expression was identified for selected transcripts. CONCLUSIONS:Consistent differences in DNA methylation between IBD cases and controls at regulatory sites within these genes suggest that their altered transcription contributes to IBD pathogenesis.

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Is theabundanceof Faecalibacteriumprausnitzii relevanttoCrohn’s disease? Wenjing Jia, Rebekah N. Whitehead, Lesley Griffiths, Claire Dawson, Rosemary H. Waring, David B. Ramsden, John O. Hunter & Jeffrey A. Cole School of Biosciences, University of Birmingham, Birmingham, UK; Gastroenterology Research Unit, Addenbrookes Hospital, Cambridge, UK; and Medical School, University of Birmingham, Birmingham, UK

Introduction As one of the possible causes of inflammatory bowel disease (IBD), the microbiota in the human gut has been widely studied. Sulphate-reducing bacteria (SRB) have long been considered as candidate pathogens because their metabolic product, sulphide, through its toxic properties, damages the colonic epithelium. However, no specific SRB has been identified as a pathogen to IBD, and even their abundance in the human gut remains controversial. This project compares the amount of faecal SRB in healthy subjects with that in patients either with Crohn's disease (CD) or ulcerative colitis (UC), and traces the change, if any, after treatment in each patient. Methods Faecal samples were collected from 18 healthy subjects, 18 CD patients and 14 UC patients. CD patients were treated with elemental diet and UC patients received conventional treatment. A suspension of the faecal matter from each patient both before and after treatment was inoculated into a sulphate-rich growth medium, Postgate B. The abundance of SRB was gauged according to the amount of black precipitate (ferrous sulphide) formed by SRB. Results Contrary to expectation 88% of healthy subjects carry SRB that produce high levels of sulphide in vitro, despite its cytotoxicity in vivo. In contrast, this carriage rate is low (45%) in CD patients at their initial presentation. After taking elemental diet, the carriage rate of active SRB did not change despite a significant fall in the total amount of gut bacteria. In UC patients, who are often characterised with mucus depletion, the carriage rate of active SRB is significantly lower (31%, p<0.05) when compared to healthy control. This finding is consistent with a previous proposal that sulphate in mucins rather than that in undigested food is the energy source for SRB. After conventional treatment, the carriage rate increased from 31% to 69%. Conclusion In healthy subjects, SRB that produce high levels of sulphide in vitro are prevalent in the human gut but do not cause adverse effects. An impaired mucous sulphation might be associated with most of UC patients who carry low levels of active SRB.

Reports that bacteria within the Firmicutes phylum, especially the species Faecalibacterium prausnitzii, are less abundant in Crohn's disease (CD) patients and supernatants from cultures of this bacterium are anti-inflammatory prompted the investigation of the possible correlations between the abundance of F. prausnitzii and the response to treatment in patients with gut diseases and healthy controls. In a randomized, double-blind trial, faeces were collected from healthy volunteers, and from patients with active CD, ulcerative colitis (UC) and irritable bowel syndrome before and after treatment. The levels of F. prausnitzii DNA in faecal suspensions were determined by PCR. Treatment by an elemental diet was effective, resulting in decreases in both the Harvey and Bradshaw index (P <0.001) and the concentrations of serum C-reactive protein (P <0.05). The total levels of F. prausnitzii in faecal samples from CD patients at presentation were lower than those in the other groups both before and after the treatment. There was no correlation between F. prausnitzii abundance and the severity of CD before treatment. Clinical improvement unexpectedly correlated with a significant decrease in the abundance of F. prausnitzii, especially the A2-165 subgroup (P <0.05). Our data suggest that a paucity of F. prausnitzii in the gastrointestinal microbial communities is likely to be a minor aetiological factor in CD: recovery following elemental diet is attributed to lower levels of gut flora.

Background: Identification of Crohn's disease (CD)-associated genetic variants is key to understanding pathogenic pathways underlying disease susceptibility. Recent reports of an association between TNFSF15 variants and CD have been modestly replicated in European populations, suggesting heterogeneity at this locus with stronger CD association in Japanese than European populations.Methods: We investigated the association between variants in TNFSF15 and CD in 756 CD patients and 636 controls. Disease subphenotype associations were also investigated.Results: TNFSF15 single nucleotide polymorphism (SNP) variants were associated with CD in our panel with peak odds ratio (OR) 1.2 (95% confidence interval [CI] 1.01-1.41) P = 0.033. The presence of a risk haplotype was replicated for the first time in a European population (frequency 67% in cases and 61% in controls) OR = 1.44 (95% CI 1.23-1.68) P = 0.00012. This result mirrors the UK panel in the index study (Yamazaki et a] [2005] Hum Mol Genet 14:3499-3506) but is less significant than that reported in Japanese populations. There was no evidence of association with any individual CD subphenotype.Conclusions: Variants in TNFSF15 contribute to overall CD susceptibility in European populations, although to a lesser extent than that seen in the Japanese. Further studies to define the precise disease-causing variants as well as targeted functional studies are now required in human CD as TNFSF15 is a potential target for biological therapies.