LARGE SINGLE-CENTER STUDY CONFIRMS KNOWN NUDT15, TPMT, AND HLA ASSOCIATIONS WITH THIOPURINE-ASSOCIATED ADVERSE EVENTS AND REVEALS PLEIOTROPIC EFFECTS AT THESE LOCI FOR THIOPURINE ADVERSE DRUG REACTIONS.

Introduction: We aimed to investigate the relationship between consumption of ultra-processed foods (UPFs) and: 1) active symptomatic disease and 2) intestinal inflammation in a cohort of adults living with inflammatory bowel disease (IBD).

Methods: Participant data (n=141) was used from the prospective Manitoba Living with IBD study. Food intake was assessed using the Harvard Food Frequency Questionnaire (FFQ). UPF consumption was determined at 1-year follow-up using the NOVA Classification system. The percentage of total energy consumption from UPFs (NOVA4) was calculated and divided into 3 tertiles. We used linear regression analysis to assess the association between UPFs (using low [T1] vs. high [T3] consumption of UPFs) and active disease using the IBD Symptom Inventory (IBDSI) score of >14 for CD and >13 UC; and intestinal inflammation as measured by a fecal calprotectin level of >250 ug/g.

Results: The mean number of episodes of active symptomatic disease over one year was significantly higher among persons with UC, but not CD, in the higher consumption of UPFs group (T3) compared to the lower consumption of UPF (T1) group (13.9 vs 5.8, p=0.037). There were no significant differences in the mean number of episodes of inflammation over one year for lower vs. upper consumption of UPF in either UC or CD persons. When adjusting for age, gender, disease type and disease duration, the number of episodes of active symptomatic disease was 8 times less for the lower consumption of UPF (T1) compared to the higher consumption of UPF (T3) (Beta= -8.42, p=0.01) among UC.

Conclusion: UPF consumption appears to be a predictor of active symptomatic disease. While it was not significantly associated with increased inflammation, the study limitation of having fewer points of measurement in the year for FCAL may have underpowered this finding. Reducing UPF consumption is an additional dietary strategy that can be suggested as a means of minimizing symptomatic disease among people living with IBD.

Background:
The pathogenesis of Crohn’s disease (CD) involves complex interactions between host genetics, environment, and the intestinal microbiome. Bile acids (BAs) can act as signaling molecules involved in host immune regulation, and potentially in CD pathogenesis. Primary BAs help absorb dietary fat and are bio transformed into secondary BAs by the gut microbiome. However, the relationship between BAs, dietary fat, and CD development is unknown. We aimed to investigate the relationship between CD onset, BAs, and dietary fat in a pre-disease setting, and evaluate the predictive performance of these factors on CD onset via machine learning models.

Methods: Healthy first-degree relatives of CD patients were recruited as part of the Crohn’s and Colitis Canada Genetic Environment Microbial (GEM) project. In a nested case-control sub-cohort, 87 subjects diagnosed with CD on follow up were matched 1:4 to subjects who remained unaffected, by age, sex, follow-up time, and geographic location (n=347). Baseline serum, urine, and stool BAs were measured with the combination of four ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy. We used recruitment food frequency questionnaire data, deriving dietary fat types with the 2015 StatsCan Nutrient database. We used Generalized Estimation Equations to explore relationships between BAs (n=92) and dietary fat (n=9), and conditional logistic regressions to identify associations between BAs and dietary fats with CD. Finally, we used a tree-based machine-learning algorithm (XGBoost) with 5-fold cross-validation to assess the predictive performance of BA and dietary fat on future onset of CD. Two-sided p<0.05 defined significance.

Results:
In total, 22 BAs associated with dietary fat (p<0.05). Next, alterations in serum-derived cholate, glycocholate glucuronide, and glycoursodeoxycholic acid sulfate, stool-derived lithocholate, urine-derived deoxycholic acid 12-sulfate, and monounsaturated fat intake were associated with increased odds of CD (p<0.05). For CD onset prediction, serum-derived BAs had the best predictive performance (mean AUC of 0.70 [95% CI: 0.63-0.76]), followed by stool derived BAs (mean AUC= 0.61 [0.50-0.71]), and urine derived BAs (mean AUC= 0.52 [0.43-0.61]). In contrast, dietary fats were not predictive of CD onset (mean AUC= 0.49 [0.39-0.62]).

Conclusions: Currently, serum-derived BAs better predict the risk of CD than stool or urine derived BA, while dietary fat is not predictive of CD risk. BAs may play a role in the pathogenesis of CD, years before diagnosis.

Funding
The Leona M. and Harry B. Helmsley Charitable Trust
Kenneth Croitoru received the Canada Research Chair in Inflammatory Bowel Diseases
The International Organization for the Study of Inflammatory Bowel Diseases (IOIBD)
Jingcheng Shao received the Data Science Institute Summer Undergraduate Data Science award

Background: The pathogenesis of inflammatory bowel disease (IBD) which includes Crohn’s disease (CD) and ulcerative colitis (UC), is believed to involve activation of the intestinal immune system in response to the gut microbiome among genetically susceptible hosts. Several medications have been considered to contribute to the etiology of IBD. This study assessed the association between medication use and risk of developing IBD using the Prospective Urban Rural Epidemiology (PURE) cohort.

Methods: This was a prospective cohort study of 133,137 individuals between the ages of 20-80 from 24 countries. Country-specific validated questionnaires documented baseline and follow-up medication use. Participants were followed prospectively at least every 3 years. The main outcome was development of IBD, including Crohn’s disease (CD) and ulcerative colitis (UC). Short-term (baseline but not follow-up use) and long-term use (baseline and subsequent follow-up use) was evaluated. Results are presented as adjusted odds ratios (aOR) with 95% confidence intervals (CI).

Results: During the median follow-up of 11.0 years [interquartile range (IQR) 9.2-12.2], we recorded 571 incident cases of IBD (143 CD and 428 UC). Higher risk of incident IBD was associated with baseline antibiotic use [aOR: 2.81 (95% CI: 1.67-4.73), p=0.0001] and hormonal medication use [aOR: 4.43 (95% CI: 1.78-11.01), p=0.001]. Among females, previous or current oral contraceptive use was also associated with IBD development [aOR: 2.17 (95% CI: 1.70-2.77), p=5.02E-10]. NSAID users were also observed to have increased risk of IBD [aOR: 1.80 (95% CI: 1.23-2.64), p=0.002], which was driven by long-term users [aOR: 5.58 (95% CI: 2.26-13.80), p<0.001]. All significant results were consistent in direction for CD and UC with low heterogeneity.

Conclusion: Antibiotics, hormonal medications, oral contraceptives, and long-term NSAID use were associated with increased odds of incident IBD after adjustment for covariates.

Background: Thiopurines are widely used to treat inflammatory bowel disease (IBD) and other immune mediated diseases but can cause serious toxicity. Studies have revealed that variants in thiopurine methyltransferase (TPMT) and nudix hydrolase 15 (NUDT15) are associated with thiopurine-induced leukopenia, and HLA-DQA1-HLA-DRB1 mutations confer susceptibility to thiopurine-induced pancreatitis. We aimed to further replicate these signals and also identify novel genetic markers associated with thiopurine toxicity in patients with IBD.
Methods: Whole exome sequencing was performed using Illumina platform and GATK best-practice variant calling and QC were performed following standard pipelines. Clinical data were identified via retrospective chart review of patients with IBD at a single tertiary IBD clinical center from 2005 to 2022. . Genetic association was assessed by Firth Regression in Plink2 with Principal Components for population stratification analyses included.
Results: 2687 thiopurines exposure cases were included. Adverse events to thiopurines were reported in 750/2687 (27.9%) cases including leukopenia (n=157, 5.84%), pancreatitis (104, 3.87%), hepatotocixity (213, 7.93%) and bone marrow toxicity (187, 6.96%)(these included the leukopenia subjects). Despite the fact that it is routine in our practice to ‘screen’ TPMT prior to starting thiopurines we still observed an association between leukopenia and genetic variation in TPMT (OR 2.19, P = 0.018). We also observed that TPMT variation was associated with thiopurine-induced hepatitis (OR 6.91, P = 8.89 e^-4). Thiopurine induced leukopenia was associated with variation in NUDT15 (OR 27.57, P = 0.01). NUDT15 variants were also associated with drug-induced pancreatitis (OR 4.36, P = 2.41e^-4). We also replicated the previously reported association between HLA-DQA1 (OR 3.65, P = 9.36e^-5) and HLA-DRB1 (OR 2.43, P = 3.18e^-4) and thiopurine-induced pancreatitis. HLA-DRB1 was also associated with the development of leukopenia (OR 2.92, P = 7.17e^-4) and thiopurine-induced hepatitis (OR 7.06, P =8.46e^-4). We observed several putative additional associations at HLA and the rest of the genome but none of these achieved genome-wide significance.
Conclusion: Among patients with IBD we confirmed previous associations between thiopurine-induced adverse events with TPMPT, NUDT15 (leukopenia) and HLA (pancreatitis). Our findings suggest pleiotropic effects at these loci with associations observed beyond those established by previous studies. Our findings provide additional evidence in support of recommendations to screen for these variants prior to starting a thiopurine. On-going studies include refining the risk of these variants across diverse populations as well as fine-mapping additional HLA associations and replicating our putative loci in independent cohorts.