Publications

 

Xue M, Lee SH, Shao J, Leibovitzh H, Huynh HQ, Griffiths AM, Turner D, Madsen KL, Moayyedi P, Steinhart AH, Silverberg MS, Deslandres C, Bitton A, Mack DR, Jacobson K, Ropeleski MJ, Cino M, Aumais G, Bernstein CN, Panaccione R, Bressler B, Espin-Garcia O, Xu W, Turpin W, Croitoru K; Crohn’s and Colitis Canada Genetics Environment Microbial Project Research Consortium. Metabolomics reveal distinct molecular pathways associated with future risk of Crohn's Disease. Gut Microbes 2025 Dec;17(1):2546998[CB1] . 

 

Host - microbiome interactions are central to Crohn's disease pathogenesis; yet the early metabolic alterations that precede disease onset remain poorly defined. To explore preclinical metabolic signatures of Crohn's disease, we analyzed baseline serum metabolomic profiles in a nested case-control study within the Crohn's and Colitis Canada - Genetics, Environment, Microbiome (CCC-GEM) Project, a prospective cohort of 5,122 healthy first-degree relatives of Crohn's disease patients. We included 78 individuals who later developed Crohn's disease and 311 matched first-degree relatives who remained disease-free. In an untargeted assessment of metabolomic data, we identified 63 metabolites significantly associated with future Crohn's disease risk. Integrative analyses further identified multiple associations between Crohn's disease-related metabolites and proteomic markers, gut microbiome composition, antimicrobial antibody, fecal calprotectin and C-reactive protein. Quinolinate, a tryptophan catabolite, was elevated in individuals who later developed Crohn's disease and showed strong positive correlations with C-reactive protein, fecal calprotectin, and C-X-C motif chemokine ligand 9 (CXCL9).In contrast, higher levels of ascorbate and isocitrate were associated with reduced Crohn's disease risk and were negatively correlated with C-reactive protein and Crohn's disease -associated proteins. These findings identify several distinct molecular pathways that contribute to Crohn's disease pathogenesis.

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Lee SH, Bushra M, Qui L, Griffiths AM, Turpin W, Croitoru K.and the Crohn’s and Colitis Canada-Genetic, Environmental, Microbial (CCC-GEM) Project Research Consortium. Early life exposure to parental Crohn’s disease is associated with offspring’s gut microbiome, gut permeability, and increased risk of future Crohn’s disease. Gastroenterology 2025; 168: 385-388.

 

1,252 offspring of persons with CD were enrolled in which 586 (46.8%) offspring had exposure to parental CD during the perinatal period, and 666 (53.2%) had exposure to parental CD diagnosis after the perinatal period. 14 (1.11%) individuals developed CD, of whom 11 (0.88%) were exposed to parental CD perinatally Participants exposed to a parent’s CD diagnosis during the perinatal period had a 4.31-fold higher risk (95% CI, 1.28–17.46) of developing CD compared to those exposed to a parent who developed CD later in life  In the subgroup of participants with exposure to a mother with CD, we noted a trend toward increased risk (ie, higher HR) of incident CD with earlier exposure to mother’s CD diagnosis. Offspring whose mothers developed CD more than 1 year before birth (ie, before conception) had a 6.3-fold higher risk of developing CD compared to those whose mothers developed CD later in life. Participants exposed to mother’s CD diagnosis within 1 year before birth had a 5.5-fold higher risk of incident CD, those exposed up to 1 year after birth had a 4.7-fold higher risk, and those exposed up to 3 years after birth had a 3.8-fold higher risk However, we did not find this trend in the subgroup exposed to father’s CD diagnosis.

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Lee SH, Turpin W, Espin-Garcia O, Xu W, Croitoru K; Crohn's and Colitis Canada-Genetic, Environmental, Microbial (CCC-GEM) Project Research Consortium. Development and Validation of an Integrative Risk Score for Future Risk of Crohn's Disease in Healthy First-Degree Relatives: A Multicenter Prospective Cohort Study. Gastroenterology 2025 Jan;168(1):150-153.

 

Despite advances in medical therapy, approximately one-half of patients with Crohn’s disease develop complications ultimately requiring surgery. Currently, no treatment offers a chance of cure. The increasing incidence and prevalence of Crohn’s disease globally pose a substantial burden at the population and individual levels. Thus, a better understanding of the early triggers of Crohn’s disease is desperately needed to enhance early detection, improve medical therapy, and offer a possibility of interventions that prevent development of Crohn’s disease. Previous studies have reported candidate predisease biomarkers associated with future development of Crohn’s disease, supporting the concept of a preclinical phase. However, these studies lack integration of individual predisease signatures and the multidimensional mechanisms of disease pathogenesis. To date, no risk-stratification model has combined multiple predictive biomarkers to predict future risk of developing Crohn’s disease in healthy at-risk individuals.

In this study, we leveraged a large prospective multicenter cohort of healthy first-degree relatives of people with Crohn’s disease —the Crohn’s and Colitis Canada-Genetic, Environmental, Microbial (GEM) study. Using available demographic characteristics, physiologic biomarkers, and fecal microbiome data, we developed an integrative multidimensional risk model—the GEM integrative risk score (IRS)—that predicts an first-degree relatives future risk of developing Crohn’s disease. We subsequently validated the performance of GEM-IRS in 2 testing cohorts.

We included participants with a minimum 3 months of follow-up duration as of February 28, 2021; participants recruited from countries with sufficient median follow-up duration of at least 5 years (ie, Canada, United States, and Israel). Fecal microbial profiling was performed by 16S ribosomal RNA sequencing; microbial function was imputed using PICRUSt2. Fecal calprotectin (FCP) was measured as a biomarker of gut inflammation and intestinal barrier function was assessed by the ratio of the urinary fractional excretion of lactulose over that of mannitol (LMR).

After quality control of microbiome data and removal of missing data, there were 2619 participants (1170 North American training set, 1141 North American test set, and 308 from Israel), which we used for subsequent analyses. During a median follow-up duration of 6.8 years (interquartile range 4.2–9.6 years), 61 of 2619 participants (2.3%) developed Crohn’s disease.

The GEM-IRS, built with random survival forest, resulted in a predictive performance measured by a Harrell’s concordance index of 0.951 (95% CI, 0.925–0.976) in the North American training cohort. On validation, the GEM-IRS had a concordance index of 0.789 (95% CI, 0.713–0.865) in the pooled testing cohort. Of note, the performance was higher compared with the other models tested (ie, 0.777 for gradient boosting survival and 0.717 for penalized Cox proportional hazards model).

The cumulative incidence of CD in the 2 testing cohorts was assessed using the quartiles of the GEM-IRS from the training cohort. The fourth quartile compared with the lower 3 quartiles showed a significantly increased risk of Crohn’s disease onset (hazard ratio, 6.42; 95% CI, 3.10–13.30) in the pooled testing cohort.

 

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Olivera PA, Martinez-Lozano H, Leibovitzh H, Xue M, Neustaeter A, Espin-Garcia O, Xu W, Madsen KL, Guttman DS, Bernstein CN, Yerushalmi B, Hyams JS, Abreu MT, Marshall JK, Wrobel I, Mack DR, Jacobson K, Bitton A, Aumais G, Panacionne R, Dieleman LA, Silverberg MS, Steinhart AH, Moayyedi P, Turner D, Griffiths AM, Turpin W, Lee SH, Croitoru K; Crohn’s and Colitis Canada (CCC) Genetic, Environmental, Microbial (GEM) Project Research Consortium. Healthy first-degree relatives from multiplex families versus simplex families have a higher subclinical intestinal inflammation, a distinct fecal microbial signature, and harbor a higher risk of developing Crohn's disease. Gastroenterology 2025; 168 (1):99-110.

 

Background & aims: Unaffected first-degree relatives from families with at least 2 affected first-degree relatives with Crohn's disease (multiplex families) have a high risk of developing Crohn’s disease, although the underlying mechanisms driving this risk are poorly understood. We aimed to identify differences in biomarkers between first-degree relatives from multiplex vs simplex families and investigate the risk of future Crohn’s disease onset accounting for potential confounders.

Methods: We assessed the Crohn's and Colitis Canada Genetic Environmental Microbial (GEM) cohort of healthy first-degree relatives of patients with Crohn’s disease. Genome-wide Crohn’s disease-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 Crohn’s disease multiplex status and baseline biomarkers were determined using generalized estimating equations models. Cox models were used to assess the risk of future Crohn’s disease onset.

Results: There were 4051 participants from simplex families and 334 from Crohn’s disease multiplex families. Crohn’s disease multiplex status was significantly associated with higher baseline FCP (P = .026) but not with baseline Crohn’s disease-polygenic risk scores or the lactulose-to-mannitol ratio. Three bacterial genera were found to be differentially abundant between both groups. Crohn’s disease multiplex status at recruitment was independently associated with an increased risk of developing Crohn’s disease (adjusted hazard ratio, 3.65; 95% confidence interval, 2.18-6.11, P < .001).

Conclusion: Within first-degree relatives of patients with Crohn’s disease, participants from multiplex families had a 3-fold increased risk of Crohn’s disease 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 first-degree relatves from multiplex families.

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Xue M, Leibovitzh H, Neustaeter A, Dong M, Xu W, Espin-Garcia O, Griffiths AM, Steinhart AH, Turner D, Huynh H, Dieleman LA, Panaccione R, Aumais G, Bressler B, Bitton A, Murthy S, Marshall JK, Hyams JS, Otley A, Bernstein CN, Moayyedi P, El-Matary W, Fich A, Denson LA, Ropeleski MJ, Abreu MT, Deslandres C, Cino M,  Avni-Biron I, Lee SH, Turpin W, Croitoru K. Environmental Factors Associated with Risk of Crohn’s Disease Development in A Prospective Cohort of Healthy First-Degree Relatives of Crohn’s disease Patients. Clinical Gastroenterology and Hepatology 2024 Sep;22(9):1889-1897.

 

Background: To date, it is unclear how environmental factors influence Crohn's disease risk and how they interact with biological processes. This study investigates the association between environmental exposures and Crohn's disease risk and evaluates their association with pre-disease biomarkers.

Methods: We studied 4289 healthy first-degree relatives of patients with Crohn's disease   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; gut inflammation via fecal calprotectin levels; and fecal microbiome composition through 16S rRNA sequencing.

Results: Over a 5.62-year median follow-up, 86 first degree relatives developed Crohn's disease. 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 Crohn's disease 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 Crohn's disease risk. Furthermore, living with a dog was associated with reduced lactulose to mannitol ratio; altered relative abundance of multiple bacterial genera, and increased Chao1 diversity, whereas bird owners had higher fecal calprotectin levels. Large family during participants' first year of life was associated with altered microbiota composition without affecting fecal calprotectin or lactulose to mannitol ratio.

Conclusion: This study identifies environmental variables associated with Crohn's disease risk. These variables were also associated with altered barrier function, subclinical inflammation, and gut microbiome composition shifts, suggesting potential roles in Crohn's disease pathogenesis.

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Leibovitzh H, Lee SH, Raygoza Garay JA, Espin-Garcia O, Xue M, Neustaeter A, Goethel A, Huynh HQ, Griffiths AM, Turner D, Madsen KL, Moayyedi P, Steinhart AH, Silverberg MS, Deslandres C, Bitton A, Mack DR, Jacobson K, Cino M, Aumais G, Bernstein CN, Panaccione R, Weiss B, Halfvarson J, Xu W, Turpin W, Croitoru K; Crohn’s and Colitis Canada (CCC) Genetic, Environmental, Microbial (GEM) Project Research Consortium. Immune response and barrier dysfunction-related proteomic signatures in preclinical phase of Crohn's disease highlight earliest events of pathogenesis. Gut 2023; Aug;72(8):1462-1471.

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The measure of serum proteome in the preclinical state of Crohn's disease may provide insight into biological pathways involved in Crohn's disease pathogenesis. We aimed to assess associations of serum proteins with future Crohn's disease onset and with other biomarkers predicting Crohn's disease risk in a healthy at-risk cohort. 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 Crohn's disease (n=71) were matched with four first-degree relatives 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 Crohn’s disease 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 Crohn’s disease. C-X-C motif chemokine 9 (CXCL9) had the highest odds ratio with future risk of Crohn’s disease (Odds ratio=2.07 per SD, 95% CI 1.58 to 2.73, q=7.9e-5), whereas matrix extracellular phosphoglycoprotein had the lowest odds ratio (Odds ratio 0.44, 95% CI 0.29 to 0.66, q=0.02). Notably, CXCL9 was the only analyte significantly associated with all other Crohn’s disease-risk biomarkers with consistent direction of effect (FCP: Odds ratio=2.21; LMR: Odds ratio=1.67; AS: Odds ratio=1.59) (q<0.05 for all). Conclusion: We identified serum proteomic signatures associated with future Crohn’s disease development, reflecting potential early biological processes of immune and barrier dysfunction.

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Leibovitzh H, Lee SH, Xue M, Raygoza Garay JA, Hernandez-Rocha C, Madsen KL, Meddings JB, Guttman DS, Espin-Garcia O, Smith M, Goethel M, Griffiths AM, Moayyedi P, Steinhart AH, Panancionne R, Huynh H, Jacobson K, Aumais G, Mack D, Abreu M, Bernstein CN, Marshall JK, Turner D, Xu W, Turpin W, Croitoru K, on behalf of the Crohn’s and Colitis Canada Genetics Environment Microbial Project Research Consortium. Altered gut microbiome composition and function are associated with gut barrier dysfunction in healthy relatives of patients with Crohn’s disease. Gastroenterology 2022; 163:1364-1376

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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.

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Turpin W, Dong M, Sasson G, Garay JAR, Espin-Garcia O, Lee SH, Neustaeter A, Smith MI, Lebovic H, Guttman DS, Griffiths AM, Huynh H, Dieleman L, Panaccione R, Steinhart AH, Silverberg MS, Aumais G, Jacobson K, Mack D, Murthy S, Marshall JK, Bernstein CN, Abreu M, Moayyedi P, Xu W, Paterson AD, The CCC GEM Project Research Consortium, Croitoru K. Mediterranean-like dietary pattern associations with gut microbiome composition and sub-clinical gastrointestinal inflammation.Gastroenterology 2022; 163: 685-698.

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Case-control studies have shown that patients with Crohn's disease have a microbial composition different from healthy individuals. Although the causes of Crohn's disease 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 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 of patients with Crohn's disease. As part of the Genetic, Environmental, Microbial (GEM) Project, we recruited a cohort of 2289 healthy first-degree relatives of patients with Crohn's disease. 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.

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Raygoza Garay JA, Turpin W, Lee SH, Smith MI, Goethel A, Griffiths AM, Moayyedi P, Espin-Garcia O, Abreu M, Aumais GL, Bernstein CN, Biron IA, Cino M, Deslandres C, Dotan I, El-Matary W, Feagan B, Guttman DS, Huynh H, Dieleman LA, Hyams JS, Jacobson K, Mack D, Marshall JK, Otley A, Panaccione R, Ropeleski M, Silverberg MS, Steinhart AH, Turner D, Yerushalmi B, Paterson AD, Xu W; CCC GEM Project Research Consortium; Croitoru K. Gut Microbiome Composition Is Associated With Future Onset of Crohn's Disease in Healthy First-Degree Relatives. Gastroenterology 2023;165(3):670-681.

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The cause of Crohn's disease 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 Crohn's disease 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 Crohn's disease or is the result of inflammation or drug treatment. In this prospective cohort study, 3483 healthy first-degree relatives of patients with Crohn's disease 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 Crohn's disease. 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 Crohn's disease. The performance of the model was assessed in an independent validation cohort. Results: In the validation cohort, the microbiome risk score model yielded a hazard ratio of 2.24 (95% confidence interval, 1.03-4.84; P = .04), using the median of the microbiome risk score from the discovery cohort as the threshold. The microbiome risk score 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.

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Turpin W, Lee SH, Raygoza Garay JA, Madsen KL, Meddings JB, Bedrani L, Power N, Espin-Garcia O, Xu W, Smith MI, Griffiths AM, Moayyedi P, Turner D, Seidman EG, Steinhart HA, Marshall JK, Jacobson K, Mack D, Huynh H, Bernstein CN, Paterson AD, Croitoru K; CCC GEM Project Research Consortium. Increased Intestinal Permeability is Associated with Later Development of Crohn's Disease.  Gastroenterology 2020; 159;2092-2100. 

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This study was from the Crohns and Colitis Canada sponsored GEM study. Increased intestinal permeability has been associated with Crohn's disease, 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 Crohn's disease. We assessed the intestinal permeability, measured by the urinary fractional excretion of lactulose-to-mannitol ratio at recruitment in 1420 asymptomatic first-degree relatives (6-35 years old) of patients with Crohn's disease (collected from 2008 through 2015). Participants were then followed up for a diagnosis of Crohn's disease from 2008 to 2017, with a median follow-up time of 7.8 years. We analyzed data from 50 participants who developed Crohn's disease 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 Crohn's disease based on the baseline lactulose-to-mannitol ratio. An abnormal lactulose-to-mannitol ratio (>0.03) was associated with a diagnosis of Crohn's disease 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 Crohn's disease (hazard ratio, 1.62; 95% CI, 1.051-2.50; P = .029).

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We concluded that 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.