Human induced pluripotent stem cells (hiPSCs) derived cells reflect tissue specificity found in patients with Leigh syndrome French Canadian variant (LSFC).

Leigh syndrome French Canadian type (LSFC) is a recessive neurodegenerative disease characterized by tissue-specific deficiency in cytochrome c oxidase (COX), the fourth complex in the oxidative phosphorylation system. LSFC is caused by mutations in the leucine rich pentatricopeptide repeat containing gene (LRPPRC). Most LSFC patients in Quebec are homozygous for an A354V substitution that causes a decrease in the expression of the LRPPRC protein. While LRPPRC is ubiquitously expressed and is involved in multiple cellular functions, tissue-specific expression of LRPPRC and COX activity is correlated with clinical features. In this proof-of-principle study, we developed human induced pluripotent stem cell (hiPSC)-based models from fibroblasts taken from a patient with LSFC, homozygous for the LRPPRC*354V allele, and from a control, homozygous for the LRPPRC*A354 allele. Specifically, for both of these fibroblast lines we generated hiPSC, hiPSC-derived cardiomyocytes (hiPSC-CMs) and hepatocyte-like cell (hiPSC-HLCs) lines, as well as the three germ layers. We observed that LRPPRC protein expression is reduced in all cell lines/layers derived from LSFC patient compared to control cells, with a reduction ranging from ∼70% in hiPSC-CMs to undetectable levels in hiPSC-HLC, reflecting tissue heterogeneity observed in patient tissues. We next performed exploratory analyses of these cell lines and observed that COX protein expression was reduced in all cell lines derived from LSFC patient compared to control cells. We also observed that mutant LRPPRC was associated with altered expression of key markers of endoplasmic reticulum stress response in hiPSC-HLCs but not in other cell types that were tested. While this demonstrates feasibility of the approach to experimentally study genotype-based differences that have tissue-specific impacts, this study will need to be extended to a larger number of patients and controls to not only validate the current observations but also to delve more deeply in the pathogenic mechanisms of LSFC.

Comprehensive Association Analyses of Extraintestinal Manifestations in Inflammatory Bowel Disease.

BACKGROUND & AIMS: Patients with inflammatory bowel disease (IBD) frequently develop extraintestinal manifestations (EIMs) that contribute substantially to morbidity. We assembled the largest multicohort data set to date to investigate the clinical, serologic, and genetic factors associated with EIM complications in IBD. METHODS: Data were available in 12,083 unrelated European ancestry IBD cases with presence or absence of EIMs (eg, ankylosing spondylitis [ankylosing spondylitis and sacroiliitis], primary sclerosing cholangitis [PSC], peripheral arthritis, and skin and ocular manifestations) across 4 cohorts (Cedars-Sinai Medical Center, National Institute for Diabetes and Digestive and Kidney Diseases IBD Genetics Consortium, Sinai Helmsley Alliance for Research Excellence Consortium, and Risk Stratification and Identification of Immunogenetic and Microbial Markers of Rapid Disease Progression in Children with Crohn's Disease cohort). Clinical and serologic parameters were analyzed by means of univariable and multivariable regression analyses using a mixed-effects model. Within-case logistic regression was performed to assess genetic associations. RESULTS: Most EIMs occurred more commonly in female subjects (overall EIM: P = 9.0E-05, odds ratio [OR], 1.2; 95% CI, 1.1-1.4), with CD (especially colonic disease location; P = 9.8E-09, OR, 1.7; 95% CI, 1.4-2.0), and in subjects who required surgery (both CD and UC; P = 3.6E-19, OR, 1.7; 95% CI, 1.5-1.9). Smoking increased risk of EIMs except for PSC, where there was a "protective" effect. Multiple serologic associations were observed, including with PSC (IgG and IgA, perinuclear anti-nuclear cytoplasmic antibody; anti-Saccharomyces cerevisiae antibodies; and anti-flagellin) and any EIM (IgG and IgA, perinuclear anti-nuclear cytoplasmic antibody; anti-Saccharomyces cerevisiae antibodies; and anti-Pseudomonas fluorescens-associated sequence). We identified genome-wide significant associations within major histocompatibility complex (ankylosing spondylitis and sacroiliitis, P = 1.4E-15; OR, 2.5; 95% CI, 2.0-3.1; PSC, P = 2.7E-10; OR, 2.8; 95% CI, 2.0-3.8; ocular, P = 2E-08, OR, 3.6; 95% CI, 2.3-5.6; and overall EIM, P = 8.4E-09; OR, 2.2; 95% CI, 1.7-2.9) and CPEB4 (skin, P = 2.7E-08; OR, 1.5; 95% CI, 1.3-1.8). Genetic associations implicated tumor necrosis factor, JAK-STAT, and IL6 as potential targets for EIMs. Contrary to previous reports, only 2% of our subjects had multiple EIMs and most co-occurrences were negatively correlated. CONCLUSIONS: We have identified demographic, clinical, and genetic associations with EIMs that revealed underlying mechanisms and implicated novel and existing drug targets-important steps toward a more personalized approach to IBD management.

Gene-metabolite annotation with shortest reactional distance enhances metabolite genome-wide association studies results.

Metabolite genome-wide association studies (mGWAS) have advanced our understanding of the genetic control of metabolite levels. However, interpreting these associations remains challenging due to a lack of tools to annotate gene-metabolite pairs beyond the use of conservative statistical significance threshold. Here, we introduce the shortest reactional distance (SRD) metric, drawing from the comprehensive KEGG database, to enhance the biological interpretation of mGWAS results. We applied this approach to three independent mGWAS, including a case study on sickle cell disease patients. Our analysis reveals an enrichment of small SRD values in reported mGWAS pairs, with SRD values significantly correlating with mGWAS p values, even beyond the standard conservative thresholds. We demonstrate the utility of SRD annotation in identifying potential false negatives and inaccuracies within current metabolic pathway databases. Our findings highlight the SRD metric as an objective, quantitative and easy-to-compute annotation for gene-metabolite pairs, suitable to integrate statistical evidence to biological networks.

Clinical predictors of early and late endoscopic recurrence following ileocolonic resection in Crohn's disease.

BACKGROUND AND AIM: Multiple factors are suggested to place Crohn's disease patients at risk of recurrence after ileocolic resection with conflicting associations. We aimed to identify clinical predictors of recurrence at first (early) and further (late) postoperative colonoscopy. METHODS: Crohn's disease patients undergoing ileocolic resection were prospectively recruited at six North American centers. Clinical data was collected and endoscopic recurrence was defined as Rutgeerts score ≥i2. A multivariable model was fitted to analyze variables independently associated with recurrence. RESULTS: A total of 365 patients undergoing 674 postoperative colonoscopies were included with a median age of 32 years, 189 (51.8%) were male and 37 (10.1%) non-Whites. Postoperatively, 133 (36.4%) used anti-TNF and 30 (8.2%) were smokers. At first colonoscopy, 109 (29.9%) had recurrence. Male gender (OR = 1.95, 95% CI 1.12 - 3.40), non-White ethnicity (OR = 2.48, 95% CI 1.09 - 5.63), longer interval between surgery and colonoscopy (OR = 1.09, 95% CI 1.002 - 1.18), and postoperative smoking (OR = 2.78, 95% CI 1.16 - 6.67) were associated with recurrence, while prophylactic anti-TNF reduced the risk (OR = 0.28, 95% CI 0.14 - 0.55). Postoperative anti-TNF prophylaxis had a protective effect on anti-TNF experienced patients but not on anti-TNF naïve patients. Among patients without recurrence at first colonoscopy, Rutgeerts score i1 was associated with subsequent recurrence (OR = 4.43, 95% CI 1.73 - 11.35). CONCLUSIONS: We identified independent clinical predictors of early and late Crohn's disease postoperative endoscopic recurrence. Clinical factors traditionally used for risk stratification failed to predict recurrence and need to be revised.

The role of admixture in the rare variant contribution to inflammatory bowel disease.

BACKGROUND: Identification of rare variants involved in complex, polygenic diseases like Crohn's disease (CD) has accelerated with the introduction of whole exome/genome sequencing association studies. Rare variants can be used in both diagnostic and therapeutic assessments; however, since they are likely to be restricted to specific ancestry groups, their contributions to risk assessment need to be evaluated outside the discovery population. Prior studies implied that the three known rare variants in NOD2 are absent in West African and Asian populations and only contribute in African Americans via admixture. METHODS: Whole genome sequencing (WGS) data from 3418 African American individuals, 1774 inflammatory bowel disease (IBD) cases, and 1644 controls were used to assess odds ratios and allele frequencies (AF), as well as haplotype-specific ancestral origins of European-derived CD variants discovered in a large exome-wide association study. Local and global ancestry was performed to assess the contribution of admixture to IBD contrasting European and African American cohorts. RESULTS: Twenty-five rare variants associated with CD in European discovery cohorts are typically five-fold lower frequency in African Americans. Correspondingly, where comparisons could be made, the rare variants were found to have a predicted four-fold reduced burden for IBD in African Americans, when compared to European individuals. Almost all of the rare CD European variants were found on European haplotypes in the African American cohort, implying that they contribute to disease risk in African Americans primarily due to recent admixture. In addition, proportion of European ancestry correlates the number of rare CD European variants each African American individual carry, as well as their polygenic risk of disease. Similar findings were observed for 23 mutations affecting 10 other common complex diseases for which the rare variants were discovered in European cohorts. CONCLUSIONS: European-derived Crohn's disease rare variants are even more rare in African Americans and contribute to disease risk mainly due to admixture, which needs to be accounted for when performing cross-ancestry genetic assessments.

Investigating the Crime Scene-Molecular Signatures in Inflammatory Bowel Disease.

Inflammatory bowel diseases (IBD) are without cure and troublesome to manage because of the considerable diversity between patients and the lack of reliable biomarkers. Several studies have demonstrated that diet, gut microbiota, genetics and other patient factors are essential for disease occurrence and progression. Understanding the link between these factors is crucial for identifying molecular signatures that identify biomarkers to advance the management of IBD. Recent technological breakthroughs and data integration have fuelled the intensity of this research. This research demonstrates that the effect of diet depends on patient factors and gut microbial activity. It also identifies a range of potential biomarkers for IBD management, including mucosa-derived cytokines, gasdermins and neutrophil extracellular traps, all of which need further evaluation before clinical translation. This review provides an update on cutting-edge research in IBD that aims to improve disease management and patient quality of life.

Genetic coding variant in complement factor B (CFB) is associated with increased risk for perianal Crohn's disease and leads to impaired CFB cleavage and phagocytosis.

OBJECTIVE: Perianal Crohn's disease (pCD) occurs in up to 40% of patients with CD and is associated with poor quality of life, limited treatment responses and poorly understood aetiology. We performed a genetic association study comparing CD subjects with and without perianal disease and subsequently performed functional follow-up studies for a pCD associated SNP in Complement Factor B (CFB). DESIGN: Immunochip-based meta-analysis on 4056 pCD and 11 088 patients with CD from three independent cohorts was performed. Serological and clinical variables were analysed by regression analyses. Risk allele of rs4151651 was introduced into human CFB plasmid by site-directed mutagenesis. Binding of recombinant G252 or S252 CFB to C3b and its cleavage was determined in cell-free assays. Macrophage phagocytosis in presence of recombinant CFB or serum from CFB risk, or protective CD or healthy subjects was assessed by flow cytometry. RESULTS: Perianal complications were associated with colonic involvement, OmpC and ASCA serology, and serology quartile sum score. We identified a genetic association for pCD (rs4151651), a non-synonymous SNP (G252S) in CFB, in all three cohorts. Recombinant S252 CFB had reduced binding to C3b, its cleavage was impaired, and complement-driven phagocytosis and cytokine secretion were reduced compared with G252 CFB. Serine 252 generates a de novo glycosylation site in CFB. Serum from homozygous risk patients displayed significantly decreased macrophage phagocytosis compared with non-risk serum. CONCLUSION: pCD-associated rs4151651 in CFB is a loss-of-function mutation that impairs its cleavage, activation of alternative complement pathway, and pathogen phagocytosis thus implicating the alternative complement pathway and CFB in pCD aetiology.

Identifying high-impact variants and genes in exomes of Ashkenazi Jewish inflammatory bowel disease patients.

Inflammatory bowel disease (IBD) is a group of chronic digestive tract inflammatory conditions whose genetic etiology is still poorly understood. The incidence of IBD is particularly high among Ashkenazi Jews. Here, we identify 8 novel and plausible IBD-causing genes from the exomes of 4453 genetically identified Ashkenazi Jewish IBD cases (1734) and controls (2719). Various biological pathway analyses are performed, along with bulk and single-cell RNA sequencing, to demonstrate the likely physiological relatedness of the novel genes to IBD. Importantly, we demonstrate that the rare and high impact genetic architecture of Ashkenazi Jewish adult IBD displays significant overlap with very early onset-IBD genetics. Moreover, by performing biobank phenome-wide analyses, we find that IBD genes have pleiotropic effects that involve other immune responses. Finally, we show that polygenic risk score analyses based on genome-wide high impact variants have high power to predict IBD susceptibility.

Gene-metabolite annotation with shortest reactional distance enhances metabolite genome-wide association studies results.

Studies combining metabolomics and genetics, known as metabolite genome-wide association studies (mGWAS), have provided valuable insights into our understanding of the genetic control of metabolite levels. However, the biological interpretation of these associations remains challenging due to a lack of existing tools to annotate mGWAS gene-metabolite pairs beyond the use of conservative statistical significance threshold. Here, we computed the shortest reactional distance (SRD) based on the curated knowledge of the KEGG database to explore its utility in enhancing the biological interpretation of results from three independent mGWAS, including a case study on sickle cell disease patients. Results show that, in reported mGWAS pairs, there is an excess of small SRD values and that SRD values and p-values significantly correlate, even beyond the standard conservative thresholds. The added-value of SRD annotation is shown for identification of potential false negative hits, exemplified by the finding of gene-metabolite associations with SRD ≤1 that did not reach standard genome-wide significance cut-off. The wider use of this statistic as an mGWAS annotation would prevent the exclusion of biologically relevant associations and can also identify errors or gaps in current metabolic pathway databases. Our findings highlight the SRD metric as an objective, quantitative and easy-to-compute annotation for gene-metabolite pairs that can be used to integrate statistical evidence to biological networks.

Serum Lipidomic Screen Identifies Key Metabolites, Pathways, and Disease Classifiers in Crohn's Disease.

BACKGROUND: There is an unmet medical need for biomarkers that capture host and environmental contributions in inflammatory bowel diseases (IBDs). This study aimed at testing the potential of circulating lipids as disease classifiers given their major roles in inflammation. METHODS: We applied a previously validated comprehensive high-resolution liquid chromatography-mass spectrometry-based untargeted lipidomic workflow covering 25 lipid subclasses to serum samples from 100 Crohn's disease (CD) patients and 100 matched control subjects. Findings were replicated and expanded in another 200 CD patients and 200 control subjects. Key metabolites were tested for associations with disease behavior and location, and classification models were built and validated. Their association with disease activity was tested using an independent cohort of 42 CD patients. RESULTS: We identified >70 metabolites with strong association (P < 1 × 10-4, q < 5 × 10-4) to CD. Highly performing classification models (area under the curve > 0.84-0.97) could be built with as few as 5 to 9 different metabolites, representing 6 major correlated lipid clusters. These classifiers included a phosphatidylethanolamine ether (O-16:0/20:4), a sphingomyelin (d18:1/21:0) and a cholesterol ester (14:1), a very long-chain dicarboxylic acid [28:1(OH)] and sitosterol sulfate. These classifiers and correlated lipids indicate a dysregulated metabolism in host cells, notably in peroxisomes, as well as dysbiosis, oxidative stress, compromised inflammation resolution, or intestinal membrane integrity. A subset of these were associated with disease behavior or location. CONCLUSIONS: Untargeted lipidomic analyses uncovered perturbations in the circulating human CD lipidome, likely resulting from multiple pathogenic mechanisms. Models using as few as 5 biomarkers had strong disease classifier characteristics, supporting their potential use in diagnosis or prognosis.

This study reports a comprehensive untargeted lipidomic analysis of 600 serum samples from patients with Crohn's disease and matched control subjects, identified and replicated ~70 metabolites associated with Crohn's disease, and developed highly performing classification models (area under the curve > 0.84-0.97) with as few as 5 metabolites.

Identifying transcript-level differential expression in primary human immune cells.

BACKGROUND: Multipotential hematopoietic stem cells differentiate into a wide variety of immune cells with a diversity of functions, including the ability to respond to a variety of stimuli. Importantly, numerous studies have demonstrated the importance of gene transcription in defining cell identity and functions. While these studies have primarily been performed at the level of the gene, it is known that key immune genes such as CD44 and CD45 generate multiple different transcripts that are differentially expressed across different immune cells, and that encode proteins with different sequences and functions. Prior genomic surveys have shown that the mechanisms for generating diversity in expressed transcripts (alternate splicing, alternate transcription start sites, etc.) are very active in immune cells, but have been lacking in terms of identifying genes with multiple transcripts, that are differentially expressed, and likely to affect cell functions. METHODS: We first identified the set of genes that had at least two transcripts expressed in our RNA sequencing dataset generated from purified populations of neutrophils, monocytes and five lymphocyte populations (B, NK, γδ T, CD4 + T and CD8 + T) from twelve healthy donors. Next, we developed a heuristic approach to identify genes where two or more transcripts have distinct expression patterns across lymphoid and/or myeloid populations. We then focused our annotation and interpretation on differentially expressed transcripts that affect the coding sequence. This process was repeated to identify transcripts that were differentially expressed between monocytes and populations of macrophages and LPS-stimulated macrophages derived from these monocytes in vitro. RESULTS: We found that over 55 % of genes had two or more expressed transcripts, with an average ∼3 transcripts per gene, and that 70 % of these had at least two of the transcripts that encoded proteins with different sequences. As expected, we identified a complex pattern of differential expression for multiple transcripts encoding the CD45 transmembrane protein, but we also found similar evidence for ten other genes (CD300A, FYB1, GPI, LITAF, PSMA1, PTMA, RPL32, SEPTIN9, SH3BP2, SH3KBP1) when comparing the expression patterns of transcripts within myeloid and lymphoid cells. We also identified five genes with differentially expressed transcripts associated with the transition from monocytes to macrophages (FNBP1, KLF6, and SEPTIN9) or between macrophages and LPS-stimulated macrophages (CD44, OAZ2, and SEPTIN9). For the most part, we found that the different transcripts of these genes are expected to impact specific biological functions, for example the different transcripts of SEPTIN9 likely regulate the cytoskeleton in immune cells via their interactions with actins filaments and microtubules. CONCLUSIONS: This analytic approach successfully identified multi-transcript genes that are differentially expressed across immune cells and could be applied to other transcriptomic data. DATA AVAILABILITY STATEMENT: Researchers can request access to the individual-level data from the current study by contacting the Montreal Heart Institute ethics committee at the following institutional email address: cer.icm@icm-mhi.org.

Prostaglandins and calprotectin are genetically and functionally linked to the Inflammatory Bowel Diseases.

BACKGROUND: Genome wide association studies (GWAS) have identified and validated more than 200 genomic loci associated with the inflammatory bowel disease (IBD), although for most the causal gene remains unknown. Given the importance of myeloid cells in IBD pathogenesis, the current study aimed to uncover the role of genes within IBD genetic loci that are endogenously expressed in this cell lineage. METHODS: The open reading frames (ORF) of 42 genes from IBD-associated loci were expressed via lentiviral transfer in the THP-1 model of human monocytes and the impact of each of these on the cell's transcriptome was analyzed using a RNA sequencing-based approach. We used a combination of genetic and pharmacologic approaches to validate our findings in the THP-1 line with further validation in human induced pluripotent stem cell (hiPSC)-derived-monocytes. RESULTS: This functional genomics screen provided evidence that genes in four IBD GWAS loci (PTGIR, ZBTB40, SLC39A11 and NFKB1) are involved in controlling S100A8 and S100A9 gene expression, which encode the two subunits of calprotectin (CP). We demonstrated that increasing PTGIR expression and/or stimulating PTGIR signaling resulted in increased CP expression in THP-1. This was further validated in hiPSC-derived monocytes. Conversely, knocking-down PTGIR endogenous expression and/or inhibiting PTGIR signaling led to decreased CP expression. These analyses were extended to the known IBD gene PTGER4, whereby its specific agonist also led to increased CP expression. Furthermore, we demonstrated that the PTGIR and PTGER4 mediated control of CP expression was dependent on signaling via adenylate cyclase and STAT3. Finally, we demonstrated that LPS-mediated increases in CP expression could be potentiated by agonists of PTGIR and PTGER4, and diminished by their antagonists. CONCLUSION: Our results support a causal role for the PTGIR, PTGER4, ZBTB40, SLC39A11 and NFKB1 genes in IBD, with all five genes regulating the expression of CP in myeloid cells, as well as potential roles for the prostacyclin/prostaglandin biogenesis and signaling pathways in IBD susceptibility and pathogenesis.

Epigenetic reader SP140 loss of function drives Crohn's disease due to uncontrolled macrophage topoisomerases.

How mis-regulated chromatin directly impacts human immune disorders is poorly understood. Speckled Protein 140 (SP140) is an immune-restricted PHD and bromodomain-containing epigenetic "reader," and SP140 loss-of-function mutations associate with Crohn's disease (CD), multiple sclerosis (MS), and chronic lymphocytic leukemia (CLL). However, the relevance of these mutations and mechanisms underlying SP140-driven pathogenicity remains unexplored. Using a global proteomic strategy, we identified SP140 as a repressor of topoisomerases (TOPs) that maintains heterochromatin and macrophage fate. In humans and mice, SP140 loss resulted in unleashed TOP activity, de-repression of developmentally silenced genes, and ultimately defective microbe-inducible macrophage transcriptional programs and bacterial killing that drive intestinal pathology. Pharmacological inhibition of TOP1/2 rescued these defects. Furthermore, exacerbated colitis was restored with TOP1/2 inhibitors in Sp140-/- mice, but not wild-type mice, in vivo. Collectively, we identify SP140 as a TOP repressor and reveal repurposing of TOP inhibition to reverse immune diseases driven by SP140 loss.

Human enteric viruses autonomously shape inflammatory bowel disease phenotype through divergent innate immunomodulation.

Altered enteric microorganisms in concert with host genetics shape inflammatory bowel disease (IBD) phenotypes. However, insight is limited to bacteria and fungi. We found that eukaryotic viruses and bacteriophages (collectively, the virome), enriched from non-IBD, noninflamed human colon resections, actively elicited atypical anti-inflammatory innate immune programs. Conversely, ulcerative colitis or Crohn's disease colon resection viromes provoked inflammation, which was successfully dampened by non-IBD viromes. The IBD colon tissue virome was perturbed, including an increase in the enterovirus B species of eukaryotic picornaviruses, not previously detected in fecal virome studies. Mice humanized with non-IBD colon tissue viromes were protected from intestinal inflammation, whereas IBD virome mice exhibited exacerbated inflammation in a nucleic acid sensing-dependent fashion. Furthermore, there were detrimental consequences for IBD patient-derived intestinal epithelial cells bearing loss-of-function mutations within virus sensor MDA5 when exposed to viromes. Our results demonstrate that innate recognition of IBD or non-IBD human viromes autonomously influences intestinal homeostasis and disease phenotypes. Thus, perturbations in the intestinal virome, or an altered ability to sense the virome due to genetic variation, contribute to the induction of IBD. Harnessing the virome may offer therapeutic and biomarker potential.

IBD-associated G protein-coupled receptor 65 variant compromises signalling and impairs key functions involved in inflammation.

BACKGROUND AND AIMS: Inflammatory bowel diseases (IBD) result in chronic inflammation of the gastrointestinal tract. Genetic studies have shown that the GPR65 gene, as well as its missense coding variant, GPR65*Ile231Leu, is associated with IBD. We aimed to define the signalling and biological pathways downstream of GPR65 activation and evaluate the impact of GPR65*231Leu on these. METHODS: We used HEK 293 cells stably expressing GPR65 and deficient for either Gαs, Gαq/11 or Gα12/13, to define GPR65 signalling pathways, IBD patient biopsies and a panel of human tissues, primary immune cells and cell lines to determine biologic context, and genetic modulation of human THP-1-derived macrophages to examine the impact of GPR65 in bacterial phagocytosis and NLRP3 inflammasome activation. RESULTS: We confirmed that GPR65 signals via the Gαs pathway, leading to cAMP accumulation. GPR65 can also signal via the Gα12/13 pathway leading to formation of stress fibers, actin remodeling and RhoA activation; all impaired by the IBD-associated GPR65*231Leu allele. Gene expression profiling revealed greater expression of GPR65 in biopsies from inflamed compared to non-inflamed tissues from IBD patients or control individuals, potentially explained by infiltration of inflammatory immune cells. Decreased GPR65 expression in THP-1-derived macrophages leads to impaired bacterial phagocytosis, increased NLRP3 inflammasome activation and IL-1ß secretion in response to an inflammatory stimulus. CONCLUSIONS: We demonstrate that GPR65 exerts its effects through Gαs- and Gα12/13-mediated pathways, that the IBD-associated GPR65*231Leu allele has compromised interactions with Gα12/13 and that KD of GPR65 leads to impaired bacterial phagocytosis and increased inflammatory signalling via the NLRP3 inflammasome. This work identifies a target for development of small molecule therapies.

A Role for CXCR3 Ligands as Biomarkers of Post-Operative Crohn's Disease Recurrence.

BACKGROUND AND AIMS: Crohn's disease [CD] recurrence following ileocolic resection [ICR] is common. We sought to identify blood-based biomarkers associated with CD recurrence. METHODS: CD patients undergoing ICR were recruited across six centres. Serum samples were obtained at post-operative colonoscopy. A multiplex immunoassay was used to analyse 92 inflammation-related proteins [Olink Proteomics]. Bayesian analysis was used to identify proteins associated with increasing Rutgeerts score. Identified proteins were used in receiver operating characteristic [ROC] analysis to examine the ability to identify CD recurrence [Rutgeerts score ≥i2]. Existing single cell data were interrogated to further elucidate the role of the identified proteins. RESULTS: Data from 276 colonoscopies in 213 patients were available. Median time from surgery to first and second colonoscopy was 7 (interquartile range [IQR] 6-9) and 19 [IQR 16-23] months, respectively. Disease recurrence was evident at 60 [30%] first and 36 [49%] second colonoscopies. Of 14 proteins significantly associated with Rutgeerts score, the strongest signal was seen for CXCL9 and MMP1. Among patients on anti-tumour necrosis factor drugs, CXCL9 and CXCL11 were most strongly associated with Rutgeerts score. Both are CXCR3 ligands. Incorporation of identified proteins into ROC analysis improved the ability to identify disease recurrence as compared to C-reactive protein alone: area under the curve [AUC] 0.75 (95% confidence interval [CI]: 0.66-0.82] vs 0.64 [95% CI 0.56-0.72], p = 0.012. Single cell transcriptomic data provide evidence that innate immune cells are the primary source of the identified proteins. CONCLUSIONS: CXCR3 ligands are associated with CD recurrence following ICR. Incorporation of novel blood-based candidate biomarkers may aid in identification of CD recurrence.

Serum Analyte Profiles Associated With Crohn's Disease and Disease Location.

BACKGROUND: Crohn's disease (CD) can affect any segment of the digestive tract but is most often localized in the ileal, ileocolonic, and colorectal regions of the intestines. It is believed that the chronic inflammation in CD is a result of an imbalance between the epithelial barrier, the immune system, and the intestinal microbiota. The aim of the study was to identify circulating markers associated with CD and/or disease location in CD patients. METHODS: We tested 49 cytokines, chemokines, and growth factors in serum samples from 300 patients with CD and 300 controls. After quality control, analyte levels were tested for association with CD and disease location. RESULTS: We identified 13 analytes that were higher in CD patients relative to healthy controls and that remained significant after conservative Bonferroni correction (P < 0.0015). In particular, CXCL9, CXCL1, and interleukin IL-6 had the greatest effect and were highly significant (P < 5 × 10-7). We also identified 9 analytes that were associated with disease location, with VEGF, IL-12p70, and IL-6 being elevated in patients with colorectal disease (P < 3 × 10-4). CONCLUSIONS: Multiple serum analytes are elevated in CD. These implicate the involvement of multiple cell types from the immune, epithelial, and endothelial systems, suggesting that circulating analytes reflect the inflammatory processes that are ongoing within the gut. Moreover, the identification of distinct profiles according to disease location supports the existence of a biological difference between ileal and colonic CD, consistent with previous genetic and clinical observations.

Functional screen of inflammatory bowel disease genes reveals key epithelial functions.

BACKGROUND: Genetic studies have been tremendously successful in identifying genomic regions associated with a wide variety of phenotypes, although the success of these studies in identifying causal genes, their variants, and their functional impacts has been more limited. METHODS: We identified 145 genes from IBD-associated genomic loci having endogenous expression within the intestinal epithelial cell compartment. We evaluated the impact of lentiviral transfer of the open reading frame (ORF) of these IBD genes into the HT-29 intestinal epithelial cell line via transcriptomic analyses. By comparing the genes in which expression was modulated by each ORF, as well as the functions enriched within these gene lists, we identified ORFs with shared impacts and their putative disease-relevant biological functions. RESULTS: Analysis of the transcriptomic data for cell lines expressing the ORFs for known causal genes such as HNF4a, IFIH1, and SMAD3 identified functions consistent with what is already known for these genes. These analyses also identified two major clusters of genes: Cluster 1 contained the known IBD causal genes IFIH1, SBNO2, NFKB1, and NOD2, as well as genes from other IBD loci (ZFP36L1, IRF1, GIGYF1, OTUD3, AIRE and PITX1), whereas Cluster 2 contained the known causal gene KSR1 and implicated DUSP16 from another IBD locus. Our analyses highlight how multiple IBD gene candidates can impact on epithelial structure and function, including the protection of the mucosa from intestinal microbiota, and demonstrate that DUSP16 acts a regulator of MAPK activity and contributes to mucosal defense, in part via its regulation of the polymeric immunoglobulin receptor, involved in the protection of the intestinal mucosa from enteric microbiota. CONCLUSIONS: This functional screen, based on expressing IBD genes within an appropriate cellular context, in this instance intestinal epithelial cells, resulted in changes to the cell's transcriptome that are relevant to their endogenous biological function(s). This not only helped in identifying likely causal genes within genetic loci but also provided insight into their biological functions. Furthermore, this work has highlighted the central role of intestinal epithelial cells in IBD pathophysiology, providing a scientific rationale for a drug development strategy that targets epithelial functions in addition to the current therapies targeting immune functions.

Whole-genome sequencing of African Americans implicates differential genetic architecture in inflammatory bowel disease.

Whether or not populations diverge with respect to the genetic contribution to risk of specific complex diseases is relevant to understanding the evolution of susceptibility and origins of health disparities. Here, we describe a large-scale whole-genome sequencing study of inflammatory bowel disease encompassing 1,774 affected individuals and 1,644 healthy control Americans with African ancestry (African Americans). Although no new loci for inflammatory bowel disease are discovered at genome-wide significance levels, we identify numerous instances of differential effect sizes in combination with divergent allele frequencies. For example, the major effect at PTGER4 fine maps to a single credible interval of 22 SNPs corresponding to one of four independent associations at the locus in European ancestry individuals but with an elevated odds ratio for Crohn disease in African Americans. A rare variant aggregate analysis implicates Ca2+-binding neuro-immunomodulator CALB2 in ulcerative colitis. Highly significant overall overlap of common variant risk for inflammatory bowel disease susceptibility between individuals with African and European ancestries was observed, with 41 of 241 previously known lead variants replicated and overall correlations in effect sizes of 0.68 for combined inflammatory bowel disease. Nevertheless, subtle differences influence the performance of polygenic risk scores, and we show that ancestry-appropriate weights significantly improve polygenic prediction in the highest percentiles of risk. The median amount of variance explained per locus remains the same in African and European cohorts, providing evidence for compensation of effect sizes as allele frequencies diverge, as expected under a highly polygenic model of disease.

Transethnic analysis of the human leukocyte antigen region for ulcerative colitis reveals not only shared but also ethnicity-specific disease associations.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gut. Genetic association studies have identified the highly variable human leukocyte antigen (HLA) region as the strongest susceptibility locus for IBD and specifically DRB1*01:03 as a determining factor for ulcerative colitis (UC). However, for most of the association signal such as delineation could not be made because of tight structures of linkage disequilibrium within the HLA. The aim of this study was therefore to further characterize the HLA signal using a transethnic approach. We performed a comprehensive fine mapping of single HLA alleles in UC in a cohort of 9272 individuals with African American, East Asian, Puerto Rican, Indian and Iranian descent and 40 691 previously analyzed Caucasians, additionally analyzing whole HLA haplotypes. We computationally characterized the binding of associated HLA alleles to human self-peptides and analyzed the physicochemical properties of the HLA proteins and predicted self-peptidomes. Highlighting alleles of the HLA-DRB1*15 group and their correlated HLA-DQ-DR haplotypes, we not only identified consistent associations (regarding effects directions/magnitudes) across different ethnicities but also identified population-specific signals (regarding differences in allele frequencies). We observed that DRB1*01:03 is mostly present in individuals of Western European descent and hardly present in non-Caucasian individuals. We found peptides predicted to bind to risk HLA alleles to be rich in positively charged amino acids. We conclude that the HLA plays an important role for UC susceptibility across different ethnicities. This research further implicates specific features of peptides that are predicted to bind risk and protective HLA proteins.