Leila Amininejad

Resequencing of positional candidates identifies low frequency IL23R coding variants protecting against inflammatory bowel disease

Genome-wide association studies (GWAS) have identified dozens of risk loci for many complex disorders, including Crohns disease. However, common disease-associated SNPs explain at most ∼20% of the genetic variance for Crohns disease. Several factors may account for this unexplained heritability, including rare risk variants not adequately tagged thus far in GWAS. That rare susceptibility variants indeed contribute to variation in multifactorial phenotypes has been demonstrated for colorectal cancer, plasma high-density lipoprotein cholesterol levels, blood pressure, type 1 diabetes, hypertriglyceridemia and, in the case of Crohns disease, for NOD2 (refs. 14,15). Here we describe the use of high-throughput resequencing of DNA pools to search for rare coding variants influencing susceptibility to Crohns disease in 63 GWAS-identified positional candidate genes. We identify low frequency coding variants conferring protection against inflammatory bowel disease in IL23R, but we conclude that rare coding variants in positional candidates do not make a large contribution to inherited predisposition to Crohns disease.

Common polymorphism in the PNPLA3/adiponutrin gene confers higher risk of cirrhosis and liver damage in alcoholic liver disease

BACKGROUND & AIMS A recent genome-wide association study identified genetic polymorphism (rs738409 C>G) in the PNPLA3/adiponutrin gene associated with liver steatosis. This variant has also been linked to increased risk of alcoholic liver disease (ALD) and cirrhosis in Mestizo Mexicans with excessive alcohol intake. Our aim was to study the influence of this polymorphism on European Caucasian patients with histologically suggestive ALD. METHODS Three-hundred-and-twenty-eight healthy controls and 330 ALD patients, among whom 265 had cirrhosis, were genotyped for the rs738409 polymorphism. We studied the impact of rs738409 on clinical and biological parameters, together with histological staging of steatosis and fibrosis. PNPLA3 messenger RNA (mRNA) levels were measured by quantitative real-time PCR according to the patients phenotype. RESULTS The G-allele was significantly more frequent in ALD patients than in controls (odds ratio [OR] = 1.54, 95% confidence interval [CI] = 1.12-2.11 p = 0.008) and was, among ALD patients, significantly associated with steatosis (p = 0.048), fibrosis (p = 0.001), and greater risk of cirrhosis (p = 0.001). In multivariate analysis, rs738409 remained the strongest independent factor associated with risk of cirrhosis (OR = 2.08; 95% CI = 1.15-3.77; p = 0.02). Furthermore, the PNPLA3 mRNA liver expression level was significantly lower in patients with more advanced fibrosis (p = 0.03) and negatively correlated with the hepatic venous pressure gradient (r = -0.41, p = 0.006). CONCLUSIONS In European Caucasians, the rs738409 variant is associated with increased risk of ALD, liver damage, and cirrhosis. Further prospective studies are required to confirm these results and to evaluate the potential of PNPLA3 as both a predictor and a therapeutic target in ALD.

Effect of the intake of resveratrol, resveratrol phosphate, and catechin-rich grape seed extract on markers of oxidative stress and gene expression in adult obese subjects.

Background: The preventive effect of resveratrol (RES) on the development of human diseases has been verified by numerous epidemiological studies. Resveratrol triphosphate (RTP) is a stable derivative of RES in which phosphate groups protect the phenolic groups. Aims: This study compared the effect of RTP on biochemical and molecular markers of oxidative stress to equimolar doses (0.66 mmol) of RES and catechin-rich grape seed extract (CGSE) in a model of oxidative and metabolic stress associated with obesity in humans. Methods: Thirty-two obese subjects (BMI between 30 and 40) were enrolled. They all received 1 capsule of placebo/day for 28 days before being randomly devised into three arms receiving 1 capsule/day of RES, CGSE, or RTP during the following consecutive 28 days. Blood samples were collected at baseline, after the end of placebo intake, and after the end of the investigational product intake. Biochemical parameters of oxidative stress and blood expression of 200 redox-related genes were determined at each time point. Results: RTP and CGSE showed better antioxidant activities compared to RES and induced important modulations of gene expression. Conclusion: The results suggest that RTP and CGSE could contribute to a significant reduction of oxidative stress in obese subjects.

Safety and Feasibility of Using the Second-Generation Pillcam Colon Capsule to Assess Active Colonic Crohn's Disease.

BACKGROUND & AIMS The second-generation Pillcam Colon Capsule Endoscope (PCCE-2; Given Imaging Ltd, Yoqneam, Israel) is an ingestible capsule for visualization of the colon. We performed a multicenter pilot study to assess its safety and feasibility in evaluating the severity of Crohns disease (CD). METHODS In a prospective study, 40 patients with active colonic CD underwent PCCE-2 and optical colonoscopy procedures. Using both techniques, we generated values for the Crohns Disease Endoscopic Index of Severity (CDEIS), the Simple Endoscopic Score for CD, and global evaluation of lesion severity. In the first stage of the study, we calculated the correlation between PCCE-2 and optical colonoscopy scores. In the second stage, we performed interobserver agreement analysis for a random subset of 20 PCCE-2 recordings, graded in duplicate by 2 independent readers. RESULTS There was substantial agreement between PCCE-2 and optical colonoscopy in the measurement of the CDEIS (intraclass correlation coefficient [ICC], 0.65; 95% confidence interval [CI], 0.43-0.80). There was substantial interobserver agreement between 2 independent PCCE-2 readers for the CDEIS (ICC, 0.67; 95% CI, 0.35-0.86) and the Simple Endoscopic Score for CD (ICC, 0.66; 95% CI, 0.32-0.85). However, the PCCE-2 scoring systematically underestimated the severity of disease compared with optical colonoscopy; based on our results, PCCE-2 detected colonic ulcerations with 86% sensitivity and 40% specificity. No adverse events were observed and PCCE-2 was better tolerated than colonoscopy. CONCLUSIONS PCCE-2 is feasible, safe, and well tolerated for the assessment of mucosal CD activity in selected populations. Larger studies are needed to assess its operating characteristics further. European clinical trials database number: 2014-003854-15.

Genome-wide copy number variation scan identifies complement component C4 as novel susceptibility gene for Crohn's disease

Background:The genetic component of Crohns disease (CD) is well known, with 140 susceptibility loci identified so far. In addition to single nucleotide polymorphisms typically studied in genome-wide scans, copy number variation is responsible for a large proportion of human genetic variation. Methods:We performed a genome-wide search for copy number variants associated with CD using array comparative genomic hybridization. One of the found regions was validated independently through real-time PCR. Serum levels of the found gene were measured in patients and control subjects. Results:We found copy number differences for the C4S and C4L gene variants of complement component C4 in the central major histocompatibility complex region on chromosome 6p21. Specifically, we saw that CD patients tend to have lower C4L and higher C4S copies than control subjects (P = 5.00 × 10−03 and P = 9.11 × 10−04), which was independent of known associated classical HLA I and II alleles (P = 7.68 × 10−03 and P = 6.29 × 10−03). Although C4 serum levels were not different between patients and control subjects, the relationship between C4 copy number and serum level was different for patients and control subjects with higher copy numbers leading to higher serum concentrations in control subjects, compared with CD patients (P < 0.001). Conclusions:C4 is part of the classical activation pathway of the complement system, which is important for (auto)immunity. Low C4L or high C4S copy number, and corresponding effects on C4 serum level, could lead to an exaggerated response against infections, possibly leading to (auto)immune disease.

Infliximab Trough Levels at Induction to Predict Treatment Failure During Maintenance

Background: Infliximab (IFX) is indicated for the treatment of inflammatory bowel diseases (IBD). Nevertheless, loss of response (LOR) to IFX is reported in up to 10% to 30% of patients within the first year of treatment. Our objective was to evaluate the impact of the pharmacokinetics of IFX at induction on treatment failure. Methods: This is a longitudinal cohort study on 269 patients with IBD treated with IFX in a single center. A total of 2331 blood samples were prospectively collected from 2007 until March 2015 with a retrospective analysis of clinical data. IFX trough levels (TLs) were measured by enzyme-linked immunosorbent assay. Antibodies to IFX were measured by drug-sensitive bridging assay. Results: During follow-up, patients were defined according to treatment outcome. At week 6, median IFX TL in patients requiring a switch to another treatment due to LOR (LOR switched group) (2.32 &mgr;g/mL [0.12–19.93 &mgr;g/mL]) was lower than in patients with long-term response (long-term responders) (8.66 &mgr;g/mL [0.12–12.09 &mgr;g/mL], P = 0.007) and in patients responding to optimization (LOR optimized group) (7.28 &mgr;g/mL [0.17–14.91 &mgr;g/mL], P = 0.021). At week 2, median IFX TL was lower in the LOR switched group (5.7 &mgr;g/mL [0.15–12.09 &mgr;g/mL]) compared with the long-term responders (11.92 &mgr;g/mL [0.14–19.93 &mgr;g/mL], P = 0.041) but no significant difference was reached with the LOR optimized group (11.91 &mgr;g/mL [0.23–12.09 &mgr;g/mL], P = 0.065). In the LOR switched group, median IFX TL at induction (weeks 2 and 6) was significantly lower when patients had been previously exposed to anti–tumor necrosis factor compared with naive patients (0.91 &mgr;g/mL [0.12–4.4 &mgr;g/mL] versus 6.6 &mgr;g/mL [0.15–19.93 &mgr;g/mL], P = 0.044). Conclusions: This study suggests that patients who do not respond to any optimization strategy have lower IFX TLs during induction at week 6. IFX TLs measured early on at induction might predict treatment failure to IFX during maintenance.

IBD risk loci are enriched in multigenic regulatory modules encompassing putative causative genes

GWAS have identified >200 risk loci for Inflammatory Bowel Disease (IBD). The majority of disease associations are known to be driven by regulatory variants. To identify the putative causative genes that are perturbed by these variants, we generate a large transcriptome data set (nine disease-relevant cell types) and identify 23,650 cis-eQTL. We show that these are determined by ∼9720 regulatory modules, of which ∼3000 operate in multiple tissues and ∼970 on multiple genes. We identify regulatory modules that drive the disease association for 63 of the 200 risk loci, and show that these are enriched in multigenic modules. Based on these analyses, we resequence 45 of the corresponding 100 candidate genes in 6600 Crohn disease (CD) cases and 5500 controls, and show with burden tests that they include likely causative genes. Our analyses indicate that ≥10-fold larger sample sizes will be required to demonstrate the causality of individual genes using this approach.Most of the more than 200 known genetic risk loci for inflammatory bowel disease (IBD) reside in regulatory regions. Here, the authors provide eQTL datasets for six circulating immune cell types and ileal, colonic and rectal biopsies to map regulatory modules and identify potential causative genes for IBD.

389 A Candidate Gene Study of Rare Monogenic Disorders With IBD-Like Phenotype Identified Rare Variants in XIAP Gene in a Cohort of Early-Onset IBD Patients

frameshift variant(rs5743293) had the strongest effect in Group6 (OR=5.52, p=1.07E-16) yet no effect in Group2(OR=1.12, p=0.78), with a p-value of 1.43E-7 for test of heterogeneity; the ATG16L1 T300A variant showed a strong effect in Group4(OR=0.589, p=1.68E-9), with weak to no effect observed in other groups(p for heterogeneity: 5.33E-5). An unbiased Immunochip-wide analysis with these subgroups compared to non-IBD controls identified a putative novel CD association with PRKCQ. Three PRKCQ SNPs contributed individually to different subgroups (rs113912197 to Group2, OR=24.47, p=8.18E-7; rs112123005 to Group3, OR=5.98, p=8.63E-6 and rs661985 to Group1, OR=1.34, p=4.70E-4). LD between these SNPs is weak(R2<0.1), suggesting they are independent signals. PRKCQ codes for nPKC-θ, a serine-protein kinase that mediates non-redundant functions in T-cell receptor signaling and plays an important role in the development of T-helper 2 cells following immune and inflammatory responses. PRKCQ is also a known locus for Rheumatoid Arthritis and Type I diabetes, making it functionally highly relevant. Conclusion: Using SOM, we identified 6 serologically-defined subgroups within CD. These subgroups have distinct clinical characteristics and different genetic load. Known CD loci also show dramatically different effects in these subgroups, indicating they are genetically more homogenous.

Incidence and Predictors of Success of Adalimumab Dose Escalation and De-escalation in Ulcerative Colitis: a Real-World Belgian Cohort Study

Background Adalimumab (ADM) has been shown efficacious in ulcerative colitis (UC). In randomized controlled trials, dose escalation from 40 mg ADM every other week to 40 mg every week was required in 20%-25% of patients within 1 year. Real-life data suggest higher escalation rates. Attempts for dose de-escalation have not been studied yet. We assessed the need for, outcome of, and predictors of dose escalation and de-escalation in a large retrospective cohort of UC patients treated with ADM. Methods We included 231 consecutive patients from 10 Belgian centers initiating ADM treatment for active UC before September 1, 2015 (follow-up ≥1 year in each patient). We performed detailed chart review to identify variables associated with short-term clinical benefit (based on physician global assessment and absence of rectal bleeding at week 10), success of dose escalation, and dose de-escalation. Backward Cox regression and Wald Logistic regression were used to identify predictive variables. Results Short-term clinical benefit was achieved in 101 patients (44%) and was less frequent in infliximab failures [37% vs 50%, Odds ratio 0.57 (95% CI 0.34-0.97), P = 0.038]. After a median of 2.8 (1.7-5.1) months, 164 patients (71%) needed ADM discontinuation (n = 35, 15%) or dose escalation (n = 129, 56%). Dose escalation was successful in 77/129 (60%). Dose de-escalation was attempted in 71% (55/77) after a median of 4.3 (2.9-7.2) months and was successful in 80% (43/54). Conclusions In this cohort, 56% of patients with UC required ADM dose escalation with a 60% success rate. Of note, most patients could be successfully de-escalated later on.

Trough Levels at Induction: Impact on Long Term Response when Re-Initiating Infliximab

Infliximab (IFX) is indicated for the treatment of inflammatory bowel disease (IBD) (ulcerative colitis(UC) or Crohn disease(CD)). Nevertheless, a significant proportion of patients will experience a loss of response (LOR) to IFX over time which may require despite optimization a switch to another anti-TNF or to swap out to another biotherapy. We have recently reported that week 2 and 6 IFX through levels (TLs) can be predictive of treatment failure and long term response. Only one study has shown that week 14 TLs can be predictive of long term response on re-initiation of IFX therapy. Our objective is to evaluate early on at induction IFX TLs and antibodies to IFX (ATI) in patients previously exposed to anti-TNF. 269 IBD patients (194 CD-75 UC) have been treated with IFX on follow-up. 2331 samples were prospectively collected but measured retrospectively by ELISA in parallel with clinical data. 91 samples (TL measured <1μg/ml) were analyzed for IFX ATI using drug-sensitive bridging ELISA. At follow-up, patients were subdivided into three groups: long-term responders, patients who had LOR but responded to optimization or patients who had LOR but did not respond to optimization and were switched to another biotherapy. Each group was subdivided according to naïve or previous treatment with anti-TNF (IFX or Adalimumab) status. During induction (week 2 and 6 combined), in the LOR switched group, median IFX TL was significantly lower in previously exposed patients than in naïve patients (0.92μg/ ml[0.12-4.4μg/ml]VS6.6μg/ml[0.15-19.93μg/ml], p=0.044)(Figure 1A). Inversely, there was no statistical difference between median TL in the LOR optimized group between naïve and previously exposed patients(9.38μg/ml[0.17-14.91μg/ml]vs11.82μg/ml[0.17-14.91μg/ ml], p=0.52) as well as in naïve and previously exposed Long-term responders(9.57μg/ ml[1.44-11.97μg/ml] vs 11.91μg/ml[0.12-19.93μg/ml], p=0.92). Overall, among the previously exposed patients, the LOR switched group had a lower median IFX TL (0.92μg/ ml[0.12-4.40μg/ml]) compared to the Long-term responders(9.57μg/ml[0.44-11.97μg/ml], p=0.015) and LOR optimized group(11,82μg/ml[0.23-12.09μg/ml], p=0.005)(Figure 2). The percentage of ATI occurrence was statistically lower in the Long-term responders(5.7%) than in the LOR optimized(37.5%), p= 0.002 and LOR switched groups(40%), p=0.002. Interestingly, among the LOR switched group, the percentage of ATI occurrence was similar in patients whether naïve or previously exposed to anti-TNF (38,8%VS42,9%, p= 0.86)(Figure 1B). The same observation was found in the LOR optimized group(25%VS45% p=0.45). In LOR switched group, patients previously exposed to anti-TNF seem to have lower IFX TLs at induction (at week 2 and 6) than naïve patients. This may not be related to immunogenicity as the presence of ATI was similar in patients whether naïve or previously exposed to anti-TNF.