Healthy First-Degree Relatives From Multiplex Families vs Simplex Families Have a Higher Subclinical Intestinal Inflammation, a Distinct Fecal Microbial Signature, and Harbor a Higher Risk of Developing Crohn's Disease.

BACKGROUND & AIMS: Unaffected first-degree relatives (FDRs) from families with ≥2 affected FDRs with Crohn's disease (CD, multiplex families) have a high risk of developing CD, although the underlying mechanisms driving this risk are poorly understood. We aimed to identify differences in biomarkers between FDRs from multiplex vs simplex families and investigate the risk of future CD onset accounting for potential confounders. METHODS: We assessed the Crohn's and Colitis Canada Genetic Environmental Microbial cohort of healthy FDRs of patients with CD. Genome-wide CD-polygenic risk scores, urinary fractional excretion of lactulose-to-mannitol ratio, fecal calprotectin (FCP), and fecal 16S ribosomal RNA microbiome were measured at recruitment. Associations between CD multiplex status and baseline biomarkers were determined using generalized estimating equations models. Cox models were used to assess the risk of future CD onset. RESULTS: There were 4051 participants from simplex families and 334 from CD multiplex families. CD multiplex status was significantly associated with higher baseline FCP (P = .026) but not with baseline CD-polygenic risk scores or the lactulose-to-mannitol ratio. Three bacterial genera were found to be differentially abundant between both groups. CD multiplex status at recruitment was independently associated with an increased risk of developing CD (adjusted hazard ratio, 3.65; 95% confidence interval, 2.18-6.11, P < .001). CONCLUSION: Within FDRs of patients with CD, participants from multiplex families had a 3-fold increased risk of CD onset, a higher FCP, and an altered bacterial composition, but not genetic burden or altered gut permeability. These results suggest that putative environmental factors might be enriched in FDRs from multiplex families.

Anti-Integrin αvß6 Autoantibodies Are a Novel Biomarker That Antedate Ulcerative Colitis.

BACKGROUND & AIMS: Better biomarkers for prediction of ulcerative colitis (UC) development and prognostication are needed. Anti-integrin αvß6 (anti-αvß6) autoantibodies have been described in patients with UC. We tested for the presence of anti-αvß6 antibodies in the preclinical phase of UC and studied their association with disease-related outcomes after diagnosis. METHODS: Anti-αvß6 autoantibodies were measured in 4 longitudinal serum samples collected from 82 subjects who later developed UC and 82 matched controls from a Department of Defense preclinical cohort (PREDICTS [Proteomic Evaluation and Discovery in an IBD Cohort of Tri-service Subjects]). In a distinct, external validation cohort (Crohn's and Colitis Canada Genetic Environmental Microbial project cohort), we tested 12 pre-UC subjects and 49 matched controls. Furthermore, anti-αvß6 autoantibodies were measured in 2 incident UC cohorts (COMPASS [Comprehensive Care for the Recently Diagnosed IBD Patients], n = 55 and OSCCAR [Ocean State Crohn's and Colitis Area Registry], n = 104) and associations between anti-αvß6 autoantibodies and UC-related outcomes were defined using Cox proportional hazards model. RESULTS: Anti-αvß6 autoantibodies were significantly higher among individuals who developed UC compared with controls up to 10 years before diagnosis in PREDICTS. The anti-αvß6 autoantibody seropositivity was 12.2% 10 years before diagnosis and increased to 52.4% at the time of diagnosis in subjects who developed UC compared with 2.7% in controls across the 4 time points. Anti-αvß6 autoantibodies predicted UC development with an area under the curve of at least 0.8 up to 10 years before diagnosis. The presence of anti-αvß6 autoantibodies in preclinical UC samples was validated in the GEM cohort. Finally, high anti-αvß6 autoantibodies was associated with a composite of adverse UC outcomes, including hospitalization, disease extension, colectomy, systemic steroid use, and/or escalation to biologic therapy in recently diagnosed UC. CONCLUSIONS: Anti-integrin αvß6 autoantibodies precede the clinical diagnosis of UC by up to 10 years and are associated with adverse UC-related outcomes.

Gut Microbiome Composition Is Associated With Future Onset of Crohn's Disease in Healthy First-Degree Relatives.

BACKGROUND & AIMS: The cause of Crohn's disease (CD) is unknown, but the current hypothesis is that microbial or environmental factors induce gut inflammation in genetically susceptible individuals, leading to chronic intestinal inflammation. Case-control studies of patients with CD have cataloged alterations in the gut microbiome composition; however, these studies fail to distinguish whether the altered gut microbiome composition is associated with initiation of CD or is the result of inflammation or drug treatment. METHODS: In this prospective cohort study, 3483 healthy first-degree relatives (FDRs) of patients with CD were recruited to identify the gut microbiome composition that precedes the onset of CD and to what extent this composition predicts the risk of developing CD. We applied a machine learning approach to the analysis of the gut microbiome composition (based on 16S ribosomal RNA sequencing) to define a microbial signature that associates with future development of CD. The performance of the model was assessed in an independent validation cohort. RESULTS: In the validation cohort, the microbiome risk score (MRS) model yielded a hazard ratio of 2.24 (95% confidence interval, 1.03-4.84; P = .04), using the median of the MRS from the discovery cohort as the threshold. The MRS demonstrated a temporal validity by capturing individuals that developed CD up to 5 years before disease onset (area under the curve > 0.65). The 5 most important taxa contributing to the MRS included Ruminococcus torques, Blautia, Colidextribacter, an uncultured genus-level group from Oscillospiraceae, and Roseburia. CONCLUSION: This study is the first to demonstrate that gut microbiome composition is associated with future onset of CD and suggests that gut microbiome is a contributor in the pathogenesis of CD.

Environmental Factors Associated With Risk of Crohn's Disease Development in the Crohn's and Colitis Canada - Genetic, Environmental, Microbial Project.

BACKGROUND & AIMS: To date, it is unclear how environmental factors influence Crohn's disease (CD) risk and how they interact with biological processes. This study investigates the association between environmental exposures and CD risk and evaluates their association with pre-disease biomarkers. METHODS: We studied 4289 healthy first-degree relatives (FDRs) of patients with CD from the Crohn's and Colitis Canada - Genetic, Environmental, Microbial (CCC-GEM) project. Regression models identified environmental factors associated with future CD onset and their association with pre-disease biological factors, including altered intestinal permeability measured by urinary fractional excretion of lactulose to mannitol ratio (LMR); gut inflammation via fecal calprotectin (FCP) levels; and fecal microbiome composition through 16S rRNA sequencing. RESULTS: Over a 5.62-year median follow-up, 86 FDRs developed CD. Living with a dog between ages 5 and 15 (hazard ratio [HR], 0.62; 95% confidence interval [CI], 0.40-0.96; P = .034), and living with a large family size in the first year of life (HR, 0.43; 95% CI, 0.21-0.85; P = .016) were associated with decreased CD risk, whereas having a bird at the time of recruitment (HR, 2.78; 95% CI, 1.36-5.68; P = .005) was associated with an increased CD risk. Furthermore, living with a dog was associated with reduced LMR, altered relative abundance of multiple bacterial genera, and increased Chao1 diversity, whereas bird owners had higher FCP levels. Large family during participants' first year of life was associated with altered microbiota composition without affecting FCP or LMR. CONCLUSION: This study identifies environmental variables associated with CD risk. These variables were also associated with altered barrier function, subclinical inflammation, and gut microbiome composition shifts, suggesting potential roles in CD pathogenesis.

Immune response and barrier dysfunction-related proteomic signatures in preclinical phase of Crohn's disease highlight earliest events of pathogenesis.

OBJECTIVE: The measure of serum proteome in the preclinical state of Crohn's disease (CD) may provide insight into biological pathways involved in CD pathogenesis. We aimed to assess associations of serum proteins with future CD onset and with other biomarkers predicting CD risk in a healthy at-risk cohort. DESIGN: In a nested case-control study within the Crohn's and Colitis Canada Genetics Environment Microbial Project (CCC-GEM) cohort, which prospectively follows healthy first-degree relatives (FDRs), subjects who developed CD (n=71) were matched with four FDRs remaining healthy (n=284). Using samples at recruitment, serum protein profiles using the Olink Proximity Extension Assay platform was assessed for association with future development of CD and with other baseline biomarkers as follows: serum antimicrobial antibodies (AS: positive antibody sum) (Prometheus); faecal calprotectin (FCP); gut barrier function using the fractional excretion of lactulose-to-mannitol ratio (LMR) assay. RESULTS: We identified 25 of 446 serum proteins significantly associated with future development of CD. C-X-C motif chemokine 9 (CXCL9) had the highest OR with future risk of CD (OR=2.07 per SD, 95% CI 1.58 to 2.73, q=7.9e-5), whereas matrix extracellular phosphoglycoprotein had the lowest OR (OR 0.44, 95% CI 0.29 to 0.66, q=0.02). Notably, CXCL9 was the only analyte significantly associated with all other CD-risk biomarkers with consistent direction of effect (FCP: OR=2.21; LMR: OR=1.67; AS: OR=1.59) (q<0.05 for all). CONCLUSION: We identified serum proteomic signatures associated with future CD development, reflecting potential early biological processes of immune and barrier dysfunction.

Altered Gut Microbiome Composition and Function Are Associated With Gut Barrier Dysfunction in Healthy Relatives of Patients With Crohn's Disease.

BACKGROUND & AIMS: The gut microbiome has been suggested to play a role in gut barrier hemostasis, but data are scarce and limited to animal studies. We therefore aimed to assess whether alterations in gut microbial composition and functional pathways are associated with gut barrier function in a cohort of healthy first-degree relatives of patients with Crohn's disease. METHODS: We used the Crohn's and Colitis Canada Genetic Environmental Microbial (CCC-GEM) cohort of healthy first-degree relatives of patients with Crohn's disease. Gut barrier function was assessed using the urinary fractional excretion of lactulose-to-mannitol ratio (LMR). Microbiome composition was assessed by sequencing fecal 16S ribosomal RNA. The cohort was divided into a discovery cohort (n = 2472) and a validation cohort (n = 655). A regression model was used to assess microbial associations with the LMR. A random forest classifier algorithm was performed to assess microbial community contribution to barrier function. RESULTS: Individuals with impaired barrier function (LMR >0.025) had reduced alpha-diversity (Chao1 index, P = 4.0e-4) and altered beta-diversity (Bray-Curtis dissimilarity index, R2 = 0.001, P = 1.0e-3) compared with individuals with an LMR ≤0.025. When taxa were assessed individually, we identified 8 genera and 52 microbial pathways associated with an LMR >0.025 (q < 0.05). Four genera (decreased prevalence of Adlercreutzia, Clostridia UCG 014, and Clostridium sensu stricto 1 and increased abundance of Colidextribacter) and 8 pathways (including decreased biosynthesis of glutamate, tryptophan, and threonine) were replicated in the validation cohort. The random forest approach revealed that the bacterial community is associated with gut barrier function (area under the curve, 0.63; P = 1.4e-6). CONCLUSIONS: The gut microbiome community and pathways are associated with changes in gut barrier function. These findings may identify potential microbial targets to modulate gut barrier.

Mediterranean-Like Dietary Pattern Associations With Gut Microbiome Composition and Subclinical Gastrointestinal Inflammation.

BACKGROUND & AIMS: Case-control studies have shown that patients with Crohn's disease (CD) have a microbial composition different from healthy individuals. Although the causes of CD are unknown, epidemiologic studies suggest that diet is an important contributor to CD risk, potentially via modulation of bacterial composition and gut inflammation. We hypothesized that long-term dietary clusters (DCs) are associated with gut microbiome compositions and gut inflammation. Our objectives were to identify dietary patterns and assess whether they are associated with alterations in specific gut microbial compositions and subclinical levels of gut inflammation in a cohort of healthy first-degree relatives (FDRs) of patients with CD. METHODS: As part of the Genetic, Environmental, Microbial (GEM) Project, we recruited a cohort of 2289 healthy FDRs of patients with CD. Individuals provided stool samples and answered a validated food frequency questionnaire reflecting their habitual diet during the year before sample collection. Unsupervised analysis identified 3 dietary and 3 microbial composition clusters. RESULTS: DC3, resembling the Mediterranean diet, was strongly associated with a defined microbial composition, with an increased abundance of fiber-degrading bacteria, such as Ruminococcus, as well as taxa such as Faecalibacterium. The DC3 diet was also significantly associated with lower levels of subclinical gut inflammation, defined by fecal calprotectin, compared with other dietary patterns. No significant associations were found between individual food items and fecal calprotectin, suggesting that long-term dietary patterns rather than individual food items contribute to subclinical gut inflammation. Additionally, mediation analysis demonstrated that DC3 had a direct effect on subclinical inflammation that was partially mediated by the microbiota. CONCLUSIONS: Overall, these results indicated that Mediterranean-like dietary patterns are associated with microbiome and lower intestinal inflammation. This study will help guide future dietary strategies that affect microbial composition and host gut inflammation to prevent diseases.

Novel Fecal Biomarkers That Precede Clinical Diagnosis of Ulcerative Colitis.

BACKGROUND & AIMS: Altered gut microbiota composition and function have been associated with inflammatory bowel diseases, including ulcerative colitis (UC), but the causality and mechanisms remain unknown. METHODS: We applied 16S ribosomal RNA gene sequencing, shotgun metagenomic sequencing, in vitro functional assays, and gnotobiotic colonizations to define the microbial composition and function in fecal samples obtained from a cohort of healthy individuals at risk for inflammatory bowel diseases (pre-UC) who later developed UC (post-UC) and matched healthy control individuals (HCs). RESULTS: Microbiota composition of post-UC samples was different from HC and pre-UC samples; however, functional analysis showed increased fecal proteolytic and elastase activity before UC onset. Metagenomics identified more than 22,000 gene families that were significantly different between HC, pre-UC, and post-UC samples. Of these, 237 related to proteases and peptidases, suggesting a bacterial component to the pre-UC proteolytic signature. Elastase activity inversely correlated with the relative abundance of Adlercreutzia and other potentially beneficial taxa and directly correlated with known proteolytic taxa, such as Bacteroides vulgatus. High elastase activity was confirmed in Bacteroides isolates from fecal samples. The bacterial contribution and functional significance of the proteolytic signature were investigated in germ-free adult mice and in dams colonized with HC, pre-UC, or post-UC microbiota. Mice colonized with or born from pre-UC-colonized dams developed higher fecal proteolytic activity and an inflammatory immune tone compared with HC-colonized mice. CONCLUSIONS: We have identified increased fecal proteolytic activity that precedes the clinical diagnosis of UC and associates with gut microbiota changes. This proteolytic signature may constitute a noninvasive biomarker of inflammation to monitor at-risk populations that can be targeted therapeutically with antiproteases.

Anti-Microbial Antibody Response is Associated With Future Onset of Crohn's Disease Independent of Biomarkers of Altered Gut Barrier Function, Subclinical Inflammation, and Genetic Risk.

BACKGROUND AND AIMS: Altered host immune reactivity to microbial antigens is hypothesized to trigger the onset of Crohn's disease (CD). We aimed to assess whether increased serum anti-microbial antibody response in asymptomatic first-degree relatives (FDRs) of CD patients is an independent risk factor for future CD development. METHODS: We measured host serum antibody response to 6 microbial antigens at enrollment (Prometheus enzyme-linked immunosorbent assay test: anti-Saccharomyces cerevisiae antibodies immunoglobulin A/immunoglobulin G, anti-OmpC, anti-A4-Fla2, anti-FlaX, anti-CBir1) and derived the sum of positive antibodies (AS). We used samples at enrollment of prospectively followed healthy FDRs from a nested case-control cohort of the Crohn's and Colitis Canada Genetics Environment Microbial Project. Those who later developed CD (n = 77) were matched 1:4 by age, sex, follow-up duration, and geographic location with control FDRs remaining healthy (n = 307). To address our research aims, we fitted a multivariable conditional logistic regression model and performed causal mediation analysis. RESULTS: High baseline AS (≥2) (43% of cases, 11% of controls) was associated with higher risk of developing CD (adjusted odds ratio, 6.5; 95% confidence interval, 3.4-12.7; P < .001). Importantly, this association remained significant when adjusted for markers of gut barrier function, fecal calprotectin, C-reactive protein, and CD-polygenic risk score, and in subjects recruited more than 3 years before diagnosis. Causal mediation analysis showed that the effect of high AS on future CD development is partially mediated (42%) via preclinical gut inflammation. CONCLUSIONS: Our results suggest that increased anti-microbial antibody responses are associated with risk of future development of CD, independent of biomarkers of abnormal gut barrier function, subclinical inflammation, and CD-related genetic risks. This suggests that anti-microbial antibody responses are an early predisease event in the development of CD.

Persistent Diarrhea in Patients With Crohn's Disease After Mucosal Healing Is Associated With Lower Diversity of the Intestinal Microbiome and Increased Dysbiosis.

BACKGROUND & AIMS: In patients with inflammatory bowel diseases (IBDs), symptoms do not always associate with the severity of endoscopic inflammation and can persist after mucosal healing. We investigated whether symptoms in patients with successfully treated IBD are related to the composition of the intestinal microbiome. METHODS: We analyzed 590 tissue biopsy specimens from 215 patients with IBD and 48 healthy individuals (controls). We obtained mucosal biopsy specimens from 2 colon sites (ascending and rectosigmoid) and from the terminal ileum along with clinical data. Bacterial DNA was extracted from the biopsy specimens and the V4 region of 16s ribosomal RNA sequenced by Miseq and processed using the QIIME v1.9 pipeline. RESULTS: Mucosal biopsy specimens from patients with Crohn's disease (CD) who achieved mucosal healing (Mayo scores of 0-1 or segmental endoscopic severity CD scores of 0-5) had lower Chao1 diversity than biopsy specimens from patients with ulcerative colitis (UC) or unclassified IBD (IBD-U), or controls. After endoscopic evidence of improvement in patients with UC or IBD-U, diversity of the tissue-associated microbiota did not differ significantly from that of controls. Colon biopsy specimens from patients with CD had lower microbial diversity, before and after healing (segmental endoscopic severity CD scores, 0-2), than colon biopsy specimens from controls (P < .002). In patients with CD who achieved mucosal healing, residual clinical activity (CD activity index scores >150; P = .03) and persistent diarrhea were associated with reduced microbial diversity (P = .01). Continued diarrhea was associated with a trend toward dysbiosis, based on the microbial dysbiosis index (P = .059). In patients with UC or IBD-U with moderate to severe inflammation, increasing severity of diarrhea was associated with reduced microbial diversity (P = .03). CONCLUSIONS: In an analysis of biopsy specimens from patients with IBD and controls, we found that despite endoscopic evidence of improvement or remission, α-diversity of the tissue-associated intestinal microbiome remained lower in patients with CD than in controls. This observation, along with the reduced Chao1 diversity and greater dysbiosis in intestinal microbiota of patients with residual symptoms of IBD, indicates that microbiome composition could be associated with persistent diarrhea.

Increased Intestinal Permeability Is Associated With Later Development of Crohn's Disease.

BACKGROUND & AIMS: Increased intestinal permeability has been associated with Crohn's disease (CD), but it is not clear whether it is a cause or result of the disease. We performed a prospective study to determine whether increased intestinal permeability is associated with future development of CD. METHODS: We assessed the intestinal permeability, measured by the urinary fractional excretion of lactulose-to-mannitol ratio (LMR) at recruitment in 1420 asymptomatic first-degree relatives (6-35 years old) of patients with CD (collected from 2008 through 2015). Participants were then followed up for a diagnosis of CD from 2008 to 2017, with a median follow-up time of 7.8 years. We analyzed data from 50 participants who developed CD after a median of 2.7 years during the study period, along with 1370 individuals who remained asymptomatic until October 2017. We used the Cox proportional hazards model to evaluate time-related risk of CD based on the baseline LMR. RESULTS: An abnormal LMR (>0.03) was associated with a diagnosis of CD during the follow-up period (hazard ratio, 3.03; 95% CI, 1.64-5.63; P = 3.97 × 10-4). This association remained significant even when the test was performed more than 3 years before the diagnosis of CD (hazard ratio, 1.62; 95% CI, 1.051-2.50; P = .029). CONCLUSIONS: Increased intestinal permeability is associated with later development of CD; these findings support a model in which altered intestinal barrier function contributes to pathogenesis. Abnormal gut barrier function might serve as a biomarker for risk of CD onset.

A finite mixture model for X-chromosome association with an emphasis on microbiome data analysis.

Analysis of the X chromosome has been largely neglected in genetic studies mainly because of complex underlying biological mechanisms. On the other hand, the study of human microbiome data (typically over-dispersed counts with an excess of zeros) has generated great interest recently because of advancements in next-generation sequencing technologies. We propose a novel approach to infer the association between host genetic variants in the X-chromosome and microbiome data. The method accounts for random X-chromosome inactivation (XCI), skewed (or nonrandom) XCI (XCI-S), and escape of XCI (XCI-E). The inference is performed through a finite mixture model (FMM), in which an indicator variable denoting the "true" biological mechanism is treated as missing data. An expectation-maximization algorithm on zero-inflated and two-part models is implemented to estimate genetic effects. We investigate the performance of the FMM along with strategies that assume XCI and XCI-E mechanisms for all subjects compared with alternative approaches. Briefly, an XCI mechanism codes males' genotypes as homozygous females, whereas under XCI-E, males are treated as heterozygous females. By comprehensive simulations, we evaluate tests of the hypothesis under a computationally efficient score statistic. In summary, the FMM renders reduced bias and commensurate power compared to XCI, XCI-E, and alternative strategies while maintaining adequate Type 1 error control. The proposed method has far-reaching applications. In particular, we illustrate its usage on a large-scale human microbiome study, the Genetic, Environmental and Microbial (GEM) project, to test for the genetic association on the X chromosome.

Associations of NOD2 polymorphisms with Erysipelotrichaceae in stool of in healthy first degree relatives of Crohn's disease subjects.

BACKGROUND: Genetic analyses have identified many variants associated with the risk of inflammatory bowel disease (IBD) development. Among these variants, the ones located within the NOD2 gene have the highest odds ratio of all IBD genetic risk variants. Also, patients with Crohn's disease (CD) have been shown to have an altered gut microbiome, which might be a reflection of inflammation itself or an effect of other parameters that contribute to the risk of the disease. Since NOD2 is an intracellular pattern recognition receptor that senses bacterial peptidoglycan in the cytosol and stimulates the host immune response (Al Nabhani et al., PLoS Pathog 13:e1006177, 2017), it is hypothesized that NOD2 variants represent perfect candidates for influencing host-microbiome interactions. We hypothesized that NOD2 risk variants affect the microbiome composition of healthy first degree relative (FDR) of CD patients and thus potentially contribute to an altered microbiome state before disease onset. METHODS: Based on this, we studied a large cohort of 1546 healthy FDR of CD patients and performed a focused analysis of the association of three major CD SNPs in the coding region of the NOD2 gene, which are known to confer a 15-40-fold increased risk of developing CD in homozygous or compound heterozygous individuals. RESULTS: Our results show that carriers of the C allele at rs2066845 was significantly associated with an increase in relative abundance in the fecal bacterial family Erysipelotrichaceae. CONCLUSIONS: This result suggests that NOD2 polymorphisms contribute to fecal microbiome composition in asymptomatic individuals. Whether this modulation of the microbiome influences the future development of CD remains to be assessed.