Identification of Novel Predictor Classifiers for Inflammatory Bowel Disease by Gene Expression Profiling

BACKGROUND: Improvement of patient quality of life is the ultimate goal of biomedical research, particularly when dealing with complex, chronic and debilitating conditions such as inflammatory bowel disease (IBD). This is largely dependent on receiving an accurate and rapid diagnose, an effective treatment and in the prediction and prevention of side effects and complications. The low sensitivity and specificity of current markers burden their general use in the clinical practice. New biomarkers with accurate predictive ability are needed to achieve a personalized approach that take the inter-individual differences into consideration. METHODS: We performed a high throughput approach using microarray gene expression profiling of colon pinch biopsies from IBD patients to identify predictive transcriptional signatures associated with intestinal inflammation, differential diagnosis (Crohn's disease or ulcerative colitis), response to glucocorticoids (resistance and dependence) or prognosis (need for surgery). Class prediction was performed with self-validating Prophet software package. RESULTS: Transcriptional profiling divided patients in two subgroups that associated with degree of inflammation. Class predictors were identified with predictive accuracy ranging from 67 to 100%. The expression accuracy was confirmed by real time-PCR quantification. Functional analysis of the predictor genes showed that they play a role in immune responses to bacteria (PTN, OLFM4 and LILRA2), autophagy and endocytocis processes (ATG16L1, DNAJC6, VPS26B, RABGEF1, ITSN1 and TMEM127) and glucocorticoid receptor degradation (STS and MMD2). CONCLUSIONS: We conclude that using analytical algorithms for class prediction discovery can be useful to uncover gene expression profiles and identify classifier genes with potential stratification utility of IBD patients, a major step towards personalized therapy

Análisis genómico de la enfermedad inflamatoria intestinal en tres modelos animales mediante microchips de DNA y PCR a tiempo real

Tesis Univ. Granada. Departamento de Bioquímica y Biología Molecular II. Leída el 18 de diciembre de 200

Systematic search for new predictors associated with clinical variables.

<p>Predictors were built using the tool Prophet and the 54,675 probes contained in the microarray, using the leave-one-out cross-validation strategy, to identify classifiers genes for glucocorticoid sensitivity (A), glucocorticoid dependency (B) or need for surgery (C). Data are represented as box and whiskers plots, where the error bars designate the smallest and largest observations and dots designate the outliers. Data was analyzed by two-way ANOVA followed by Bonferroni post test, <i>p</i> values higher than 0.05 were considered not significant (ns), *<i>p</i><0.01, **<i>p</i><0.001.</p

Systematic search for new predictors associated with IBD.

<p>Predictors were built using the tool Prophet and the 54,675 probes contained in the chip, using the leave-one-out cross-validation strategy, to identify classifiers genes for inflammatory bowel disease, IBD (A), Crohn’s disease, CD (B), ulcerative colitis, UC (C), CD <i>vs.</i> UC (D), low inflammation subtypes of CD and UC (E), and high inflammation subtypes of CD <i>vs.</i> UC (F). Data are represented as box and whiskers plots, where the error bars designate the smallest and largest observations and dots designate the outliers. Data was analyzed by two-way ANOVA followed by Bonferroni post test, <i>p</i> values higher than 0.05 were considered not significant (ns), *<i>p</i><0.01, **<i>p</i><0.001.</p

Discovery of Crohn’s disease (CD) and ulcerative colitis (UC) inflammatory subtypes by gene expression profiling.

<p>(A) Unsupervised hierarchical clustering of UC (blue), CD (red) and healthy controls (green) cases using the 54,675 probes contained in the chip. Up-regulated genes are shown in red and down-regulated genes in green. (B) Supervised hierarchical clustering of CD cases or UC cases using differentially expressed genes between CD and healthy (261 probes) and UC and healthy (1255 probes) respectively. This process defined two subgroups for both CD and UC cases. Functional analyses were performed using PANTHER Classification System. Examples of genes of each category are shown. (C) Clustering of samples using principal component analysis: CD1 (pink), CD2 (red), UC1 (light blue), UC2 (dark blue), healthy controls (green). (D) Association between clinical characteristics and CD and UC subtypes. Data represents the proportion of patients of each disease subtype included in different clinical variables, *<i>p</i><0.05, Fisher exact test, CD1 <i>vs.</i> CD2 or UC1 <i>vs.</i> UC2. Complete clinical data is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076235#pone.0076235.s002" target="_blank">Table S1</a>. (E) Number of differentially expressed genes (<i>p</i><0.05, t-test) in different comparisons among groups and with a fold change >1 or <−1.</p

Genomic profile of trinitrobencenesulfonic acid (TNBS)-induced colitis model (GSE9293)

[Organism] Rattus norvegicus[Experiment type] Expression profiling by array[Overall design] A single enema of 10mg of TNBS in 50% ethanol was administered to rats at day 0. Samples were recovered at days 2, 5, 7 and 14. According to inflammatory markers (myeloperoxidase activity, body weigh loss, colonic weigh/length ratio) we selected three replicates at each time point for the genimoc analysis, and 6 healthy control rats that received a saline enema. RNA was extracted from homogenized full-thickness colonic tissues in Trizol® reagent (Invitrogen) and purified with RNeasy affinity columns (Qiagen), according to manufacturer´s protocol. The microarray analysis was performed by Progenika Biopharma (Bilbao, Spain) on GeneChip® Rat Genome 230 2.0 Array (Affymetrix). All sample labeling (biotin), hybridization, staining and scanning procedures were carried out using Affimetrix, standard protocols (www.affymetrix.com). Normalization was carried out using Bioconductor sofware (affyPLM package).[Platforms] GPL1355 [Rat230_2] Affymetrix Rat Genome 230 2.0 Array[Relations] BioProject PRJNA1029331. GSM235052 Healthy control, replicate 1. [Characteristics] Sprague-Dawley female rats Tissue: full-thickness colon Rat received water2. GSM235053 Healthy control, replicate 2. Characteristics Sprague-Dawley female rats Tissue:full-thickness colon Rat received water3. GSM235055 Helathy control, replicate 3. [Characteristics] Sprague-Dawley female rats Tissue: full-thickness colon Rat received water4.GSM235056 Healthy control, replicate 4. [Characteristics] Sprague-Dawley female rats Tissue: full-thickness colon Rat received water5. GSM235057 Healthy control, replicate 5. [Characteristics] Sprague-Dawley female rats Tissue: full-thickness colon Rat received water6. GSM235058 Healthy control, replicate 6. [Characteristics] Sprague-Dawley female rats Tissue: full-thickness colon Rat received water7. GSM235552 TNBS day 2, replicate 1. [Characteristics] Sprague-Dawley female rat Tissue: full-thickness colon Rat received an enema of 10mg TNBS in 50% ethanol at day 08. GSM235558 TNBS day 2, replicate 2. [Characteristics] Sprague-Dawley female rat Tissue: full-thickness colon Rat received an enema of 10mg TNBS in 50% ethanol at day 09. GSM235559 TNBS day 2, replicate 3. [Characteristics] Sprague-Dawley female rat Tissue: full-thickness colon Rat received an enema of 10mg TNBS in 50% ethanol at day 010. GSM235643 TNBS day 5, replicate 1. [Characteristics] Sprague-Dawley female rat Tissue: full-thickness colon Rat received an enema of 10mg TNBS in 50% ethanol at day 011. GSM235745 TNBS day 5, replicate 2. [Characteristics] Sprague-Dawley female rat Tissue: full-thickness colon Rat received an enema of 10mg TNBS in 50% ethanol at day 012. GSM235832 TNBS day 5, replicate 3. [Characteristics] Sprague-Dawley female rat Tissue: full-thickness colon Rat received an enema of 10mg TNBS in 50% ethanol at day 013. GSM235872 TNBS day 7, replicate 1. [Characteristics] Sprague-Dawley female rat Tissue: full-thickness colon Rat received an enema of 10mg TNBS in 50% ethanol at day 014. GSM235873 TNBS day 7, replicate 2. [Characteristics] Sprague-Dawley female rat Tissue: full-thickness colon Rat received an enema of 10mg TNBS in 50% ethanol at day 015. GSM236126 TNBS day 7, replicate 3.[Characteristics] Sprague-Dawley female rat Tissue: full-thickness colon Rat received an enema of 10mg TNBS in 50% ethanol at day 016. GSM236922 TNBS day 14, replicate 1. [Characteristics] Sprague-Dawley female rat Tissue: full-thickness colon Rat received an enema of 10mg TNBS in 50% ethanol at day 017. GSM236924 TNBS day 14, replicate 2. [Characteristics] Sprague-Dawley female rat Tissue: full-thickness colon Rat received an enema of 10mg TNBS in 50% ethanol at day 018. GSM236925 TNBS day 14, replicate 3. [Characteristics] Sprague-Dawley female rat Tissue: full-thickness colon Rat received an enema of 10mg TNBS in 50% ethanol at day 0Trinitrobenzenesulfonic acid (TNBS) rat colitis is one of the most widely used models of inflammatory bowel disease, a condition whose etiology and pathophysiology are incompletely understood. We have characterized the model at the genomic level following a longitudinal approach