<i>In-Silico</i> Analysis of Inflammatory Bowel Disease (IBD) GWAS Loci to Novel Connections

<div><p>Genome-wide association studies (GWASs) for many complex diseases, including inflammatory bowel disease (IBD), produced hundreds of disease-associated loci—the majority of which are noncoding. The number of GWAS loci is increasing very rapidly, but the process of translating single nucleotide polymorphisms (SNPs) from these loci to genomic medicine is lagging. In this study, we investigated 4,734 variants from 152 IBD associated GWAS loci (IBD associated 152 lead noncoding SNPs identified from pooled GWAS results + 4,582 variants in strong linkage-disequilibrium (LD) (<i>r²</i> ≥0.8) for EUR population of 1K Genomes Project) using four publicly available bioinformatics tools, e.g. dbPSHP, CADD, GWAVA, and RegulomeDB, to annotate and prioritize putative regulatory variants. Of the 152 lead noncoding SNPs, around 11% are under strong negative selection (GERP++ RS ≥2); and ~30% are under balancing selection (Tajima’s D score >2) in CEU population (1K Genomes Project)—though these regions are positively selected (GERP++ RS <0) in mammalian evolution. The analysis of 4,734 variants using three integrative annotation tools produced 929 putative functional SNPs, of which 18 SNPs (from 15 GWAS loci) are in concordance with all three classifiers. These prioritized noncoding SNPs may contribute to IBD pathogenesis by dysregulating the expression of nearby genes. This study showed the usefulness of integrative annotation for prioritizing fewer functional variants from a large number of GWAS markers.</p></div

Gene interaction network using GeneMANIA webserver.

<p>Here genes are represented as nodes and edges indicate different types of interaction between genes. Black circles are the query genes and the color coding on edges indicate different types of interaction—which is defined in the network legend.</p

Top MCODE clusters from the gene interaction network.

<p>Highly connected gene clusters were identified from the network using MCODE v1.32 (a Cytoscape 2.8.2 plugin) and the top 11 clusters are presented (cutoff score ≥1).</p

List of SNPs with high GWAVA scores and HGMD-PUBLIC regulatory annotation.

<p>All the lead SNPs, as well as LD variants with GWAVA score ≥0.7 were manually screened for Human Gene Mutation Database (HGMD) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119420#pone.0119420.ref044" target="_blank">44</a>] regulatory annotation (HGMD release 2013.3) using Ensembl Genome Browser [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119420#pone.0119420.ref045" target="_blank">45</a>]. <b>TSS</b>: Transcription start site.</p><p>List of SNPs with high GWAVA scores and HGMD-PUBLIC regulatory annotation.</p

Distribution of (a) CADD and (b) GWAVA scores.

<p>Histograms are drawn taking CADD and GWAVA scores of all the variants (lead GWAS SNPs and LD variants) after removing the missing values. Here n = 4781 for CADD and n = 4460 variants for GWAVA.</p

List of prioritized putative regulatory SNPs.

<p>Position (hg19). Ref: Reference allele. Alt: Alternative allele. Cutoff: CADD ≥ 10, GWAVA ≥ 0.4 and RegulomeDB ≤ 2.</p><p>^¥ Nearby genes are obtained using <i>Ensembl Variant Effect Predictor (VEP)</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119420#pone.0119420.ref039" target="_blank">39</a>] and genes with HGNC [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119420#pone.0119420.ref040" target="_blank">40</a>] approved symbols are shown here.</p><p>List of prioritized putative regulatory SNPs.</p

Bar diagram representing the number of SNPs in each RegulomeDB category.

<p>Here n = 4734 variants. <b>ND</b>: No Data.</p

Comparing functional annotation scores of IBD associated lead noncoding GWAS SNPs versus LD variants.

<p>(<b>a</b>) CADD scores for GWAS (n = 152) versus LD variants (n = 4627). (<b>b</b>) GWAVA scores for GWAS (n = 152) versus LD variants (n = 4308). In the boxplots, center lines show the medians of the values and box limits indicate the 25th & 75th percentiles (as determined by R software). Whiskers extend to 5th and 95th percentiles and outliers are represented by open circle dots. The notches are defined as ± 1.58 × IQR (interquartile range) / square root of n and represent the 95% confidence interval for each median (default in R software). <i>P-values</i> are calculated using two-sided Wilcoxon rank sum test.</p

Selective constraints on IBD associated lead noncoding SNPs.

<p>(<b>a</b>) Evolutionary conservation versus human specific selection on lead SNPs. Evolutionary conservation is presented by GERP++ RS scores (<i>x axis</i>) and human specific selection is presented by Tajima’s D scores (<i>y axis</i>). Each point represents a single SNP; n = 148 SNPs. Positive GERP++ and TD scores indicate regions under negative selection and negative scores indicate positive selection. SNPs in the top-right segment are under strong evolutionary constraint as well as human specific selection. (<b>b</b>) Cross-population diversity of lead SNPs between CEU population and rest of the world. DAF is presented in the <i>x axis</i> and ΔDAF is presented in <i>y axis</i>. Each point represents a single SNP; n = 148 SNPs. <b>GERP++ RS</b>: Genomic Evolutionary Rate Profiling rejected substitution; <b>TD</b>: Tajima’s D; <b>DAF</b>: Derived allele frequency; <b>ΔDAF</b>: Difference in derived allele frequency; <b>CEU</b>: Utah residents with ancestry from Northern and Western Europe. TD, DAF and ΔDAF statistics are for 1K Genomes Project’s CEU population.</p

Ramadan fasting in Saudi Arabia is associated with altered expression of CLOCK, DUSP and IL-1alpha genes, as well as changes in cardiometabolic risk factors

<div><p>Background</p><p>During the fasting month of Ramadan, practicing Saudis develop severe disturbances in sleeping and feeding patterns. Concomitantly, cortisol circadian rhythm is abolished, diurnal cortisol levels are elevated and circulating levels of several adipokines are altered favouring insulin resistance.</p><p>Aim</p><p>To examine changes in the expression of CLOCK and glucocorticoid-controlled genes, such as DUSP1 and IL-1α in Saudi adults before and during Ramadan, and to investigate possible associations with selected cardiometabolic risk factors.</p><p>Methods</p><p>Healthy young volunteers (5 females, 18 males; mean age +SEM = 23.2 +1.2 years) were evaluated before Ramadan and two weeks into it. Blood samples were collected at 9 am (±1 hour) and twelve hours later for determination of serum lipid profile, high sensitivity CRP (hsCRP), and adiponectin. The expression of CLOCK, DUSP1 and IL-1α was evaluated in circulating leukocytes.</p><p>Results</p><p>Mean levels of GGT and morning adiponectin decreased, while those of LDL-c/ HDL-c and atherogenic index (AI) increased significantly in Ramadan compared to Shabaan. There was no significant difference between morning and evening adiponectin during Ramadan, while the diurnal rhythm of hsCRP was lost. CLOCK gene expression mean was significantly higher in morning than in evening during Shabaan. Mean morning and evening DUSP1 mRNA levels showed significant increase during Ramadan compared to Shabaan, however, its diurnal rhythm was maintained. Morning IL-1α mRNA expression remained significantly higher than in the evening during Ramadan, but was markedly decreased compared to Shabaan.</p><p>Discussion</p><p>Ramadan fasting in Saudi Arabia is associated with improvements in some cardiometabolic risk factors, such as circulating GGT and hsCRP and leukocyte expression of IL-1α mRNA, suggesting that intermittent fasting might have a beneficial component. These benefits may be offset by the previously reported dysregulation in the circadian rhythm, excess glucocorticoid levels and action, and insulin resistance, explaining increased prevalence of cardiometabolic disorders and type 2 diabetes mellitus.</p></div