Faecal metabolome and its determinants in inflammatory bowel disease.

OBJECTIVE: Inflammatory bowel disease (IBD) is a multifactorial immune-mediated inflammatory disease of the intestine, comprising Crohn's disease and ulcerative colitis. By characterising metabolites in faeces, combined with faecal metagenomics, host genetics and clinical characteristics, we aimed to unravel metabolic alterations in IBD. DESIGN: We measured 1684 different faecal metabolites and 8 short-chain and branched-chain fatty acids in stool samples of 424 patients with IBD and 255 non-IBD controls. Regression analyses were used to compare concentrations of metabolites between cases and controls and determine the relationship between metabolites and each participant's lifestyle, clinical characteristics and gut microbiota composition. Moreover, genome-wide association analysis was conducted on faecal metabolite levels. RESULTS: We identified over 300 molecules that were differentially abundant in the faeces of patients with IBD. The ratio between a sphingolipid and L-urobilin could discriminate between IBD and non-IBD samples (AUC=0.85). We found changes in the bile acid pool in patients with dysbiotic microbial communities and a strong association between faecal metabolome and gut microbiota. For example, the abundance of Ruminococcus gnavus was positively associated with tryptamine levels. In addition, we found 158 associations between metabolites and dietary patterns, and polymorphisms near NAT2 strongly associated with coffee metabolism. CONCLUSION: In this large-scale analysis, we identified alterations in the metabolome of patients with IBD that are independent of commonly overlooked confounders such as diet and surgical history. Considering the influence of the microbiome on faecal metabolites, our results pave the way for future interventions targeting intestinal inflammation.

Long-term dietary patterns are associated with pro-inflammatory and anti-inflammatory features of the gut microbiome.

OBJECTIVE: The microbiome directly affects the balance of pro-inflammatory and anti-inflammatory responses in the gut. As microbes thrive on dietary substrates, the question arises whether we can nourish an anti-inflammatory gut ecosystem. We aim to unravel interactions between diet, gut microbiota and their functional ability to induce intestinal inflammation. DESIGN: We investigated the relation between 173 dietary factors and the microbiome of 1425 individuals spanning four cohorts: Crohn's disease, ulcerative colitis, irritable bowel syndrome and the general population. Shotgun metagenomic sequencing was performed to profile gut microbial composition and function. Dietary intake was assessed through food frequency questionnaires. We performed unsupervised clustering to identify dietary patterns and microbial clusters. Associations between diet and microbial features were explored per cohort, followed by a meta-analysis and heterogeneity estimation. RESULTS: We identified 38 associations between dietary patterns and microbial clusters. Moreover, 61 individual foods and nutrients were associated with 61 species and 249 metabolic pathways in the meta-analysis across healthy individuals and patients with IBS, Crohn's disease and UC (false discovery rate<0.05). Processed foods and animal-derived foods were consistently associated with higher abundances of Firmicutes, Ruminococcus species of the Blautia genus and endotoxin synthesis pathways. The opposite was found for plant foods and fish, which were positively associated with short-chain fatty acid-producing commensals and pathways of nutrient metabolism. CONCLUSION: We identified dietary patterns that consistently correlate with groups of bacteria with shared functional roles in both, health and disease. Moreover, specific foods and nutrients were associated with species known to infer mucosal protection and anti-inflammatory effects. We propose microbial mechanisms through which the diet affects inflammatory responses in the gut as a rationale for future intervention studies.

Whole exome sequencing analyses reveal gene-microbiota interactions in the context of IBD.

OBJECTIVE: Both the gut microbiome and host genetics are known to play significant roles in the pathogenesis of IBD. However, the interaction between these two factors and its implications in the aetiology of IBD remain underexplored. Here, we report on the influence of host genetics on the gut microbiome in IBD. DESIGN: To evaluate the impact of host genetics on the gut microbiota of patients with IBD, we combined whole exome sequencing of the host genome and whole genome shotgun sequencing of 1464 faecal samples from 525 patients with IBD and 939 population-based controls. We followed a four-step analysis: (1) exome-wide microbial quantitative trait loci (mbQTL) analyses, (2) a targeted approach focusing on IBD-associated genomic regions and protein truncating variants (PTVs, minor allele frequency (MAF) >5%), (3) gene-based burden tests on PTVs with MAF <5% and exome copy number variations (CNVs) with site frequency <1%, (4) joint analysis of both cohorts to identify the interactions between disease and host genetics. RESULTS: We identified 12 mbQTLs, including variants in the IBD-associated genes IL17REL, MYRF, SEC16A and WDR78. For example, the decrease of the pathway acetyl-coenzyme A biosynthesis, which is involved in short chain fatty acids production, was associated with variants in the gene MYRF (false discovery rate <0.05). Changes in functional pathways involved in the metabolic potential were also observed in participants carrying rare PTVs or CNVs in CYP2D6, GPR151 and CD160 genes. These genes are known for their function in the immune system. Moreover, interaction analyses confirmed previously known IBD disease-specific mbQTLs in TNFSF15. CONCLUSION: This study highlights that both common and rare genetic variants affecting the immune system are key factors in shaping the gut microbiota in the context of IBD and pinpoints towards potential mechanisms for disease treatment.

Gut microbiota in inflammatory bowel diseases: moving from basic science to clinical applications.

In recent years, large efforts have been made to unravel the role of the gut microbiota in inflammatory bowel disease (IBD), which is a chronic inflammatory disorder of the gastro-intestinal tract. Considering the heterogeneity patients with IBD display in their disease course and response to treatment, there is a big need in translating these findings towards clinical practise. In this perspective article, we discuss strategies to facilitate the transition from basic science on gut microbiota in IBD to clinical applications. We suggest that setting gold standards, improving and increasing the biobanking efforts, and studying other members of the gut microbiota are a necessary step to reveal the exact role of the gut microbiota in IBD. In addition, we discuss the potential of the gut microbiome as a clinical tool for the diagnoses, prediction and/or treatment of the disease. We believe that the growing interest in the gut microbiota will reveal its potential in the management of IBD in a not too distant future.

Exploring the Predictive Value of Gut Microbiome Signatures for Therapy Intensification in Patients With Inflammatory Bowel Disease: A 10-Year Follow-up Study.

BACKGROUND: Inflammatory bowel diseases (IBDs) pose a significant challenge due to their diverse, often debilitating, and unpredictable clinical manifestations. The absence of prognostic tools to anticipate the future complications that require therapy intensification presents a substantial burden to patient private life and health. We aimed to explore whether the gut microbiome is a potential biomarker for future therapy intensification in a cohort of 90 IBD patients. METHODS: We conducted whole-genome metagenomics sequencing on fecal samples from these patients, allowing us to profile the taxonomic and functional composition of their gut microbiomes. Additionally, we conducted a retrospective analysis of patients' electronic records over a period of 10 years following the sample collection and classified patients into (1) those requiring and (2) not requiring therapy intensification. Therapy intensification included medication escalation, intestinal resections, or a loss of response to a biological treatment. We applied gut microbiome diversity analysis, dissimilarity assessment, differential abundance analysis, and random forest modeling to establish associations between baseline microbiome profiles and future therapy intensification. RESULTS: We identified 12 microbial species (eg, Roseburia hominis and Dialister invisus) and 16 functional pathways (eg, biosynthesis of L-citrulline and L-threonine) with significant correlations to future therapy intensifications. Random forest models using microbial species and pathways achieved areas under the curve of 0.75 and 0.72 for predicting therapy intensification. CONCLUSIONS: The gut microbiome is a potential biomarker for therapy intensification in IBD patients and personalized management strategies. Further research should validate our findings in other cohorts to enhance the generalizability of these results.

Ninety IBD patients were followed-up for 10 years after producing a fecal sample. During this period, 36% of the patients required therapy intensification. We show that the gut microbiome at baseline is associated with, and might hold predictive value for future necessity of therapy intensification.

The gut microbiome across the cardiovascular risk spectrum.

AIMS: Despite treatment advancements, cardiovascular disease remains a leading cause of death worldwide. Identifying new targets is crucial for enhancing preventive and therapeutic strategies. The gut microbiome has been associated with coronary artery disease (CAD), however our understanding of specific changes during CAD development remains limited. We aimed to investigate microbiome changes in participants without clinically manifest CAD with different cardiovascular risk levels and in patients with ST-elevation myocardial infarction (STEMI). METHODS: In this cross-sectional study, we characterized the gut microbiome using metagenomics of 411 faecal samples from individuals with low (n=130), intermediate (n=130) and high (n=125) cardiovascular risk based on the Framingham score, and STEMI patients (n=26). We analysed diversity, and differential abundance of species and functional pathways while accounting for confounders including medication and technical covariates. RESULTS: Collinsella stercoris, Flavonifractor plautii and Ruthenibacterium lactatiformans showed increased abundances with cardiovascular risk, while Streptococcus thermophilus was negatively associated. Differential abundance analysis revealed eight species and 49 predicted metabolic pathways that were differently abundant among the groups. In the gut microbiome of STEMI patients, there was a depletion of pathways linked to vitamin, lipid and amino-acid biosynthesis. CONCLUSION: We identified four microbial species showing a gradual trend in abundance from low-risk individuals to those with STEMI, and observed differential abundant species and pathways in STEMI patients compared to those without clinically manifest CAD. Further investigation is warranted to gain deeper understanding of their precise role in CAD progression and potential implications, with the ultimate goal of identifying novel therapeutic targets.

Despite previous studies demonstrating dysbiosis in STEMI patients, our understanding of the precise microbiome changes across the cardiovascular risk spectrum remains limited. This study addresses this knowledge gap by providing insights into the gut microbiome composition of individuals across varying cardiovascular risk levels and STEMI patients. By examining the gut microbiome of carefully selected participants from the general population with three different risk levels and a unique group of STEMI patients, we identified microbial species and pathways with differential abundance across the groups. Several of these species and pathways are associated with inflammation and lipid metabolism, which are key factors in CAD development. Collinsella stercoris, Flavonifractor plautii and Ruthenibacterium lactatiformans are increasingly abundant, while Streptococcus thermophilus is decreasingly abundant across the cardiovascular risk spectrum. The gut microbiome of STEMI patients showed eight differentially abundant species compared to groups at risk. Notably, four of these species, characterized by an elevated abundance in STEMI patients, have not been previously reported.

Effect of host genetics on the gut microbiome in 7,738 participants of the Dutch Microbiome Project.

Host genetics are known to influence the gut microbiome, yet their role remains poorly understood. To robustly characterize these effects, we performed a genome-wide association study of 207 taxa and 205 pathways representing microbial composition and function in 7,738 participants of the Dutch Microbiome Project. Two robust, study-wide significant (P < 1.89 × 10-10) signals near the LCT and ABO genes were found to be associated with multiple microbial taxa and pathways and were replicated in two independent cohorts. The LCT locus associations seemed modulated by lactose intake, whereas those at ABO could be explained by participant secretor status determined by their FUT2 genotype. Twenty-two other loci showed suggestive evidence (P < 5 × 10-8) of association with microbial taxa and pathways. At a more lenient threshold, the number of loci we identified strongly correlated with trait heritability, suggesting that much larger sample sizes are needed to elucidate the remaining effects of host genetics on the gut microbiome.

Gut microbiome dysbiosis is associated with increased mortality after solid organ transplantation.

Organ transplantation is a life-saving treatment for patients with end-stage disease, but survival rates after transplantation vary considerably. There is now increasing evidence that the gut microbiome is linked to the survival of patients undergoing hematopoietic cell transplant, yet little is known about the role of the gut microbiome in solid organ transplantation. We analyzed 1370 fecal samples from 415 liver and 672 renal transplant recipients using shotgun metagenomic sequencing to assess microbial taxonomy, metabolic pathways, antibiotic resistance genes, and virulence factors. To quantify taxonomic and metabolic dysbiosis, we also analyzed 1183 age-, sex-, and body mass index-matched controls from the same population. In addition, a subset of 78 renal transplant recipients was followed longitudinally from pretransplantation to 24 months after transplantation. Our data showed that both liver and kidney transplant recipients suffered from gut dysbiosis, including lower microbial diversity, increased abundance of unhealthy microbial species, decreased abundance of important metabolic pathways, and increased prevalence and diversity of antibiotic resistance genes and virulence factors. These changes were found to persist up to 20 years after transplantation. Last, we demonstrated that the use of immunosuppressive drugs was associated with the observed dysbiosis and that the extent of dysbiosis was associated with increased mortality after transplantation. This study represents a step toward potential microbiome-targeted interventions that might influence the outcomes of recipients of solid organ transplantation.

The Composition and Metabolic Potential of the Human Small Intestinal Microbiota Within the Context of Inflammatory Bowel Disease.

BACKGROUND AND AIMS: The human gastrointestinal tract harbours distinct microbial communities essential for health. Little is known about small intestinal communities, despite the small intestine playing a fundamental role in nutrient absorption and host-microbe immune homeostasis. We aimed to explore the small intestine microbial composition and metabolic potential, in the context of inflammatory bowel disease [IBD]. METHODS: Metagenomes derived from faecal samples and extensive phenotypes were collected from 57 individuals with an ileostomy or ileoanal pouch, and compared with 1178 general population and 478 IBD faecal metagenomes. Microbiome features were identified using MetaPhAn2 and HUMAnN2, and association analyses were performed using multivariate linear regression. RESULTS: Small intestinal samples had a significantly lower bacterial diversity, compared with the general population and, to a lesser extent, IBD samples. Comparing bacterial composition, small intestinal samples clustered furthest from general population samples and closest to IBD samples with intestinal resections. Veillonella atypica, Streptococcus salivarius, and Actinomyces graevenitzii were among the species significantly enriched in the small intestine. Predicted metabolic pathways in the small intestine are predominantly involved in simple carbohydrate and energy metabolism, but also suggest a higher pro-inflammatory potential. CONCLUSIONS: We described the bacterial composition and metabolic potential of the small intestinal microbiota. The colonic microbiome of IBD patients, particularly with intestinal resections, showed resemblance to that of the small intestine. Moreover, several features characterising the small intestinal microbiome have been previously associated with IBD. These results highlight the importance of studying the small intestinal microbiota to gain new insight into disease pathogenesis.

Patient attitudes towards faecal sampling for gut microbiome studies and clinical care reveal positive engagement and room for improvement.

Faecal sample collection is crucial for gut microbiome research and its clinical applications. However, while patients and healthy volunteers are routinely asked to provide stool samples, their attitudes towards sampling remain largely unknown. Here, we investigate the attitudes of 780 Dutch patients, including participants in a large Inflammatory Bowel Disease (IBD) gut microbiome cohort and population controls, in order to identify barriers to sample collection and provide recommendations for gut microbiome researchers and clinicians. We sent questionnaires to 660 IBD patients and 112 patients with other disorders who had previously been approached to participate in gut microbiome studies. We also conducted 478 brief interviews with participants in our general population cohort who had collected stool samples. Statistical analysis of the data was performed using R. 97.4% of respondents reported that they had willingly participated in stool sample collection for gut microbiome research, and most respondents (82.9%) and interviewees (95.6%) indicated willingness to participate again, with their motivations for participating being mainly altruistic (57.0%). Responses indicated that storing stool samples in the home freezer for a prolonged time was the main barrier to participation (52.6%), but clear explanations of the sampling procedures and their purpose increased participant willingness to collect and freeze samples (P = 0.046, P = 0.003). To account for participant concerns, gut microbiome researchers establishing cohorts and clinicians trying new faecal tests should provide clear instructions, explain the rationale behind their protocol, consider providing a small freezer and inform patients about study outcomes. By assessing the attitudes, motives and barriers surrounding participation in faecal sample collection, we provide important information that will contribute to the success of gut microbiome research and its near-future clinical applications.

Impact of commonly used drugs on the composition and metabolic function of the gut microbiota.

The human gut microbiota has now been associated with drug responses and efficacy, while chemical compounds present in these drugs can also impact the gut bacteria. However, drug-microbe interactions are still understudied in the clinical context, where polypharmacy and comorbidities co-occur. Here, we report relations between commonly used drugs and the gut microbiome. We performed metagenomics sequencing of faecal samples from a population cohort and two gastrointestinal disease cohorts. Differences between users and non-users were analysed per cohort, followed by a meta-analysis. While 19 of 41 drugs are found to be associated with microbial features, when controlling for the use of multiple medications, proton-pump inhibitors, metformin, antibiotics and laxatives show the strongest associations with the microbiome. We here provide evidence for extensive changes in taxonomy, metabolic potential and resistome in relation to commonly used drugs. This paves the way for future studies and has implications for current microbiome studies by demonstrating the need to correct for multiple drug use.

Gut microbial co-abundance networks show specificity in inflammatory bowel disease and obesity.

The gut microbiome is an ecosystem that involves complex interactions. Currently, our knowledge about the role of the gut microbiome in health and disease relies mainly on differential microbial abundance, and little is known about the role of microbial interactions in the context of human disease. Here, we construct and compare microbial co-abundance networks using 2,379 metagenomes from four human cohorts: an inflammatory bowel disease (IBD) cohort, an obese cohort and two population-based cohorts. We find that the strengths of 38.6% of species co-abundances and 64.3% of pathway co-abundances vary significantly between cohorts, with 113 species and 1,050 pathway co-abundances showing IBD-specific effects and 281 pathway co-abundances showing obesity-specific effects. We can also replicate these IBD microbial co-abundances in longitudinal data from the IBD cohort of the integrative human microbiome (iHMP-IBD) project. Our study identifies several key species and pathways in IBD and obesity and provides evidence that altered microbial abundances in disease can influence their co-abundance relationship, which expands our current knowledge regarding microbial dysbiosis in disease.

Anti-inflammatory Gut Microbial Pathways Are Decreased During Crohn's Disease Exacerbations.

BACKGROUND AND AIMS: Crohn's disease [CD] is a chronic inflammatory disorder of the gastrointestinal tract characterised by alternating periods of exacerbation and remission. We hypothesised that changes in the gut microbiome are associated with CD exacerbations, and therefore aimed to correlate multiple gut microbiome features to CD disease activity. METHODS: Faecal microbiome data generated using whole-genome metagenomic shotgun sequencing of 196 CD patients were of obtained from the 1000IBD cohort [one sample per patient]. Patient disease activity status at time of sampling was determined by re-assessing clinical records 3 years after faecal sample production. Faecal samples were designated as taken 'in an exacerbation' or 'in remission'. Samples taken 'in remission' were further categorised as 'before the next exacerbation' or 'after the last exacerbation', based on the exacerbation closest in time to the faecal production date. CD activity was correlated with gut microbial composition and predicted functional pathways via logistic regressions using MaAsLin software. RESULTS: In total, 105 bacterial pathways were decreased during CD exacerbation (false-discovery rate [FDR] <0.1) in comparison with the gut microbiome of patients both before and after an exacerbation. Most of these decreased pathways exert anti-inflammatory properties facilitating the biosynthesis and fermentation of various amino acids [tryptophan, methionine, and arginine], vitamins [riboflavin and thiamine], and short-chain fatty acids [SCFAs]. CONCLUSIONS: CD exacerbations are associated with a decrease in microbial genes involved in the biosynthesis of the anti-inflammatory mediators riboflavin, thiamine, and folate, and SCFAs, suggesting that increasing the intestinal abundances of these mediators might provide new treatment opportunities. These results were generated using bioinformatic analyses of cross-sectional data and need to be replicated using time-series and wet lab experiments.

SLC39A8 missense variant is associated with Crohn's disease but does not have a major impact on gut microbiome composition in healthy subjects.

BACKGROUND: Gene-microbiome interactions are important in aetiology and pathogenesis of inflammatory bowel disease, a chronic inflammatory disorder of the gastrointestinal tract consisting of Crohn's disease and ulcerative colitis. Scarce studies on gene-microbiome interactions show very little overlap in their results. Therefore, it is of utmost importance that gene-microbiome studies are repeated. We aimed to replicate the association between the SLC39A8 [Thr]391 risk allele and gut microbiome composition in patients with inflammatory bowel disease and healthy controls. METHODS: We collected faecal samples, peripheral blood and extensive phenotype data from 291 patients with inflammatory bowel disease and 476 healthy controls. Carrier status information was obtained from whole exome sequencing data, generated using the Illumina HiSeq. The gut microbiome composition was determined by tag-sequencing the 16S rRNA gene. Associations between carrier status and disease were tested using the Wilcoxon-Mann-Whitney test. Associations between carriers and gut microbiome composition were determined using principal coordinate analyses, variance explained, alpha diversity and additive general linear models in inflammatory bowel disease, healthy controls and all groups combined. RESULTS: Crohn's disease patients were more often carriers of the missense variant (21/171, 12.3%) than controls (30/476, 6.3%) (OR = 2.1, P = 0.01). We could not identify associations between carrier status and overall gut microbiome composition and microbial richness in all tested groups after correcting for potential confounding factors. We did identify 37 different operational taxonomical units to be associated with carrier status among the tested groups. Two of these 37 were identified before in the discovery study. CONCLUSIONS: We could confirm the genetic association of the SLC39A8 [Thr]391 risk allele with Crohn's disease but we could only limited replicate the association in gut microbiome composition. Independent replication of gene-microbiome studies is warranted to identify true biological mechanisms.

Analysis of 1135 gut metagenomes identifies sex-specific resistome profiles.

Several gastrointestinal diseases show a sex imbalance, although the underlying (patho)physiological mechanisms behind this are not well understood. The gut microbiome may be involved in this process, forming a complex interaction with host immune system, sex hormones, medication and other environmental factors. Here we performed sex-specific analyses of fecal microbiota composition in 1135 individuals from a population-based cohort. The overall gut microbiome composition of females and males was significantly different (p = 0.001), with females showing a greater microbial diversity (p = 0.009). After correcting for the effects of intrinsic factors, smoking, diet and medications, female hormonal factors such as the use of oral contraceptives and undergoing an ovariectomy were associated with microbial species and pathways. Females had a higher richness of antibiotic-resistance genes, with the most notable being resistance to the lincosamide nucleotidyltransferase (LNU) gene family. The higher abundance of resistance genes is consistent with the greater prescription of the Macrolide-Lincosamide-Streptogramin classes of antibiotics to females. Furthermore, we observed an increased resistance to aminoglycosides in females with self-reported irritable bowel syndrome. These results throw light upon the effects of common medications that are differentially prescribed between sexes and highlight the importance of sex-specific analysis when studying the gut microbiome and resistome.

Gut microbiota composition and functional changes in inflammatory bowel disease and irritable bowel syndrome.

Changes in the gut microbiota have been associated with two of the most common gastrointestinal diseases, inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). Here, we performed a case-control analysis using shotgun metagenomic sequencing of stool samples from 1792 individuals with IBD and IBS compared with control individuals in the general population. Despite substantial overlap between the gut microbiome of patients with IBD and IBS compared with control individuals, we were able to use gut microbiota composition differences to distinguish patients with IBD from those with IBS. By combining species-level profiles and strain-level profiles with bacterial growth rates, metabolic functions, antibiotic resistance, and virulence factor analyses, we identified key bacterial species that may be involved in two common gastrointestinal diseases.

Drug Repositioning in Inflammatory Bowel Disease Based on Genetic Information.

BACKGROUND: Currently, 200 genetic risk loci have been identified for inflammatory bowel disease (IBD). Although these findings have significantly advanced our insight into IBD biology, there has been little progress in translating this knowledge toward clinical practice, like more cost-efficient drug development. Our aim was to use genetic knowledge to identify drugs that warrant further investigation in IBD treatment. METHODS: We hypothesized that proteins encoded by IBD candidate genes are potential IBD drug targets because genetic information can increase successful drug identification. We identified drugs that target the proteins encoded by IBD candidate genes using the DrugBank. We included proteins that are in direct protein-protein interaction with proteins encoded by IBD risk genes. Promising potential IBD drugs were selected based on a manual literature search of all identified drugs (PubMed, ClinicalTrials.gov). RESULTS: We have identified 113 drugs that could potentially be used in IBD treatment. Fourteen are known IBD drugs, 48 drugs have been, or are being investigated in IBD, 19 are being used or being investigated in other inflammatory disorders treatment, and 32 are investigational new drugs that have not yet been registered for clinical use. CONCLUSIONS: We confirm that proteins encoded by IBD candidate genes are targeted by approved IBD therapies. Furthermore, we show that Food and Drug Administration-approved drugs could possibly be repositioned for IBD treatment. We also identify investigational new drugs that warrant further investigation for IBD treatment. Incorporating this process in IBD drug development will improve the utilization of genetic data and could lead to the improvement of IBD treatment.

Analysis of five chronic inflammatory diseases identifies 27 new associations and highlights disease-specific patterns at shared loci.

We simultaneously investigated the genetic landscape of ankylosing spondylitis, Crohn's disease, psoriasis, primary sclerosing cholangitis and ulcerative colitis to investigate pleiotropy and the relationship between these clinically related diseases. Using high-density genotype data from more than 86,000 individuals of European ancestry, we identified 244 independent multidisease signals, including 27 new genome-wide significant susceptibility loci and 3 unreported shared risk loci. Complex pleiotropy was supported when contrasting multidisease signals with expression data sets from human, rat and mouse together with epigenetic and expressed enhancer profiles. The comorbidities among the five immune diseases were best explained by biological pleiotropy rather than heterogeneity (a subgroup of cases genetically identical to those with another disease, possibly owing to diagnostic misclassification, molecular subtypes or excessive comorbidity). In particular, the strong comorbidity between primary sclerosing cholangitis and inflammatory bowel disease is likely the result of a unique disease, which is genetically distinct from classical inflammatory bowel disease phenotypes.