Polymorphism in the Retinoic Acid Metabolizing Enzyme CYP26B1 and the Development of Crohn’s Disease

<div><p>Several studies suggest that Vitamin A may be involved in the pathogenesis of inflammatory bowel disease (IBD), but the mechanism is still unknown. Cytochrome P450 26 B1 (CYP26B1) is involved in the degradation of retinoic acid and the polymorphism rs2241057 has an elevated catabolic function of retinoic acid, why we hypothesized that the rs2241057 polymorphism may affect the risk of Crohn’s disease (CD) and Ulcerative Colitis (UC). DNA from 1378 IBD patients, divided into 871 patients with CD and 507 with UC, and 1205 healthy controls collected at Örebro University Hospital and Karolinska University Hospital were analyzed for the <i>CYP26B1</i> rs2241057 polymorphism with TaqMan® SNP Genotyping Assay followed by allelic discrimination analysis. A higher frequency of patients homozygous for the major (T) allele was associated with CD but not UC compared to the frequency found in healthy controls. A significant association between the major allele and non-stricturing, non-penetrating phenotype was evident for CD. However, the observed associations reached borderline significance only, after correcting for multiple testing. We suggest that homozygous carriers of the major (T) allele, relative to homozygous carriers of the minor (C) allele, of the <i>CYP26B1</i> polymorphism rs2241057 may have an increased risk for the development of CD, which possibly may be due to elevated levels of retinoic acid. Our data may support the role of Vitamin A in the pathophysiology of CD, but the exact mechanisms remain to be elucidated.</p></div

Genotype frequencies of the polymorphism <i>rs2241057</i> in the <i>CYP26B1</i> gene for patients with Crohn’s disease and healthy controls, displayed for sub phenotypes and clinical features.

<p>Chi-square test used for <i>P</i>-values unless otherwise stated. Odds ratio and confidence interval estimated using 2×2 contingency tables. C =  minor allele, T =  major allele. OR =  odds ratio, CI =  95% confidence interval. ^*Patients with combination of two locations are excluded in this overview, ^† Fisher’s two tailed exact test used.</p

Allele frequencies of the polymorphism <i>rs2241057</i> in the <i>CYP26B1</i> gene for patients with Crohn’s disease versus healthy controls, displayed for sub phenotypes and clinical features.

<p>Chi-square test used for <i>P</i>-values unless otherwise stated. Odds ratio and confidence interval estimated using 2×2 contingency tables. C =  minor allele, T =  major allele. OR =  odds ratio, CI =  95% confidence interval. ^*Patients with combination of two locations are excluded in this overview, ^† Fisher’s two tailed exact test used.</p

Clinical characteristics of patients with inflammatory bowel disease.

<p>CD =  Crohn’s disease, UC =  Ulcerative colitis.</p

Genotype frequencies of the polymorphism rs2241057 in the <i>CYP26B1</i> gene in patients with inflammatory bowel disease <i>vs</i>. healthy controls.

<p>Chi-square test used for <i>P</i>-values. Odds ratio and confidence interval estimated using 2×2 contingency tables. CD =  Crohn’s disease, UC =  Ulcerative colitis, C =  minor allele, T =  major allele. OR =  odds ratio, CI =  95% confidence interval. ^* Uncorrected P-value/FDR corrected P-value.</p

Comparison of protein expression levels across disease categories.

<p>(<b>A</b>) Boxplots depicting the distribution of the fraction of the metagenomes with PSMs. Boxes indicate 25^th, 50^th and 75^th percentile, with whiskers representing 10^th and 90^th percentile points. (<b>B</b>) Gene family richness as measured by the number of KEGG Orthologous group (KO) matches in the metagenomic dataset. Grey  =  Healthy, Blue  =  CCD, Red  =  ICD.</p

Clustering of distal gut metaproteomes according to disease.

<p>Non-metric multidimensional scaling (nMDS) of distal gut metaproteomes from CD twin cohort. The different colored square symbols represent the metaproteomic profiles for each sample (Blue  =  CCD, Grey  =  Healthy, Red  =  ICD). The numbers beside the symbols refer to the specific patient ID from Dicksved et al., 2008 (proteomes were run in technical duplicates). The axes are dimensionless: the coefficients of determination for the correlations between ordination distances and distances in the original n-dimensional space are. 472 and. 831 for Axis 1 and 2, respectively. A matrix of normalized spectral counts per protein (HMRG database search) from each duplicate metaproteome was imported into PCORD v5 software. nMDS was performed using the Bray-Curtis distance measure A three-dimensional solution was found after 119 iterations. The final stress for the nMDS was 6.47458. The white spots with grey shading correspond to individual proteins identified using HMRG database. Arrows indicate strength of correlation of specific bacterial strains to ordinated data. Pearson correlation coefficients for <i>Faecalibacterium prausnitzii, Anaerofustis stercorihominis, Clostridium leptum, Bacteroides ovatus</i>, <i>Bacteroides sp. 4_3</i>, and <i>Bacteroides sp. 3_1</i> were −0.875, −0.851, 0.784, 0.8, 0.788, and 0.817, respectively.</p

Metaproteome differences between mean Healthy and mean ICD COG frequencies.

<p>To determine statistically significant differences between categories, White's non-parametric t-test was used with bootstrapping and Storey FDR multiple test correction. 95% upper and lower confidence intervals are shown. Red and grey bars indicate COG categories that are higher in ICD or Healthy metaproteomes, respectively; Asterisks indicate COG categories that were significantly different between ICD and healthy (q-value<0.05).</p

Metabolic Pathways that Differentiate Healthy and ICD phenotypes.

<p>(<b>A</b>) Metabolic pathways differentiating between healthy and ICD according to metabolic module analysis (p<0.05; 5% FDR). All pathways are less abundant in ICD compared to healthy except for <i>Bacteroides</i> membrane proteins (upper left box) that are more abundant in ICD. The colors reflect their phylogenetic origin that was determined using the lowest common ancestor of their HMRG mappings. Grey highlighted areas discussed in the main text: (1) butyrate production; (2) membrane proteins. (<b>B</b>) Observed metabolic module abundance shift versus its expected value based on the abundance of the host species. To separate out modules whose fold change is higher/lower than expected by the difference in its species abundance, we used the prediction interval of a fitted linear model (blue lines). The grey symbols are (species-separated) modules that are not significantly different between ICD and H (Wilcoxon rank-sum test; 5% FDR). They could have a high median fold change, but this is not always significant (eg when interpersonal variation is high). The colored symbols are (species-separated) modules that are significant between ICD and H (Wilcoxon rank-sum test; 5% FDR). Colored symbols inside the interval are significantly different but are in line with what would be expected from the species difference. Colored symbols outside the blue lines are higher/lower than expected. Specific <i>Faecalibacterium</i> proteins that are down regulated in the butyrate module (green squares) include the following: butyryl-CoA dehydrogenase (EC 1.3.99.2), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), enoyl-CoA hydratase/carnithine racemase, and acetyl-CoA acetyltransferases; as well as the module for lysine fermentation to acetate and butyrate (pink square). Specific <i>Bacteroides</i> proteins that are down regulated in the DNA-directed RNA polymerase module are the following (red X's): alpha and beta subunits (EC 2.7.7.6).</p

Specific genes and proteins that differ in relative amounts according to disease state.

<p>Relative Abundance of mucin-desulfating sulfatase (Mds), RagB and SusC/D, Outer Membrane Protein A (OmpA), TonB, Short-Chain Fatty Acid production (SCFA) and Butyrate production in (<b>A</b>) metagenomes and (<b>B</b>) MM metaproteomes. Error bars in (A) and (B) represent the standard error of the mean of the samples from Healthy (3 MG, 4 MP), ICD (5 MG, 6 MP) and CCD (2 MG/MP). (<b>C</b>) Specific outer membrane proteins and proteins involved in SCFA pathway that differed between disease categories. Protein abundances were calculated as normalized spectral abundance using the HMRG database search. The presence-absence heatmap indicates which of the 51 bacterial strains each protein matched to in the HMRG database search: black  =  species present, white  =  species absent. Grey  =  Healthy, Blue  =  CCD, Red  =  ICD.</p