Blood and Intestine eQTLs from an Anti-TNF-Resistant Crohn's Disease Cohort Inform IBD Genetic Association Loci

Abstract

Objectives: Genome-wide association studies (GWAS) have identified loci reproducibly associated with inflammatory bowel disease (IBD) and other immune-mediated diseases; however, the molecular mechanisms underlying most of genetic susceptibility remain undefined. Expressional quantitative trait loci (eQTL) of disease-relevant tissue can be employed in order to elucidate the genes and pathways affected by disease-specific genetic variance.

Methods: In this study, we derived eQTLs for human whole blood and intestine tissues of anti-tumor necrosis factor-resistant Crohn's disease (CD) patients. We interpreted these eQTLs in the context of published IBD GWAS hits to inform on the disease process.

Results: At 10% false discovery rate, we discovered that 5,174 genes in blood and 2,063 genes in the intestine were controlled by a nearby single-nucleotide polymorphism (SNP) (i.e., cis-eQTL), among which 1,360 were shared between the two tissues. A large fraction of the identified eQTLs were supported by the regulomeDB database, showing that the eQTLs reside in regulatory elements (odds ratio; OR=3.44 and 3.24 for blood and intestine eQTLs, respectively) as opposed to protein-coding regions. Published IBD GWAS hits as a whole were enriched for blood and intestine eQTLs (OR=2.88 and 2.05; and P value=2.51E-9 and 0.013, respectively), thereby linking genetic susceptibility to control of gene expression in these tissues. Through a systematic search, we used eQTL data to inform 109 out of 372 IBD GWAS SNPs documented in National Human Genome Research Institute catalog, and we categorized the genes influenced by eQTLs according to their functions. Many of these genes have experimentally validated roles in specific cell types contributing to intestinal inflammation.

Conclusions: The blood and intestine eQTLs described in this study represent a powerful tool to link GWAS loci to a regulatory function and thus elucidate the mechanisms underlying the genetic loci associated with IBD and related conditions. Overall, our eQTL discovery approach empirically identifies the disease-associated variants including their impact on the direction and extent of expression changes in the context of disease-relevant cellular pathways in order to infer the functional outcome of this aspect of genetic susceptibility.

Figure 1

Data analysis workflow. (a) Molecular data collection and mapping of expression Quantitative Trait Loci (eQTLs). (b) Downstream eQTLs results mining.

Figure 2

Overlap between SNP-controlled genes in blood and intestine. A large degree of overlap was observed between discovered SNP-controlled genes from blood and intestine tissues.

Figure 3

eQTLs enrichments for disease-associated loci. (a) We tested the overlap between known and validated disease/trait-associated loci (from the NHGRI GWAS catalog) and expression Quantitative Trait loci discovered from the blood and intestine of anti-TNF-resistant patients. Disease traits were sorted according to odds ratio of enrichment for disease/trait-associated loci within intestine eQTLs. In parentheses, we present the number of overlaps in the following order: number of disease/trait loci overlapping with intestine-specific eQTLs; number of disease/trait loci overlapping with eQTLs shared by blood and intestine; number of disease/trait loci overlapping with blood-specific eQTLs; total number of loci for the considered disease/trait. The heatmap color indicates the overlap log-odds ratio, with red, white, and blue indicating overlap above, in line with, and below what would be expected by random chance. (b) Intersection between IBD-associated loci (from a genome-wide analysis of the WTCCC dataset) and blood/intestine eQTLs. Highlighted some shared (green circles), blood-specific (red circle), and intestine-specific (blue circle) eQTL peaks.

Figure 4

Ingenuity pathway analysis of genes controlled by IBD-associated loci. (a) Heatmap displaying clustering of canonical pathways enriched genes (enrichment P value <0.01) whose blood expression is controlled by an IBD-associated locus (‘eGenes' henceforth); pathways on y axis and genes driving enrichment on the x axis. Dark blue squares indicate a gene is present in the corresponding pathway listed to the right. (b) Venn diagram of the intersection of Ingenuity pathways enriched in blood and intestine (refer to Supplementary Table S6 and Supplementary Table S7).

Figure 5

Cell type enrichment of blood eSNPs. Heatmap displaying clustering of cell types enriched in blood eSNPs (enrichment P value <0.05); cell types on y axis and genes driving enrichment on the x axis. Dark red squares indicate a gene is present in the corresponding pathway listed to the right.

Figure 6

Tissue specificity of IBD-associated eSNPs. Some example of eSNPs that were previously reported to be associated with IBD, with different degree of tissue specificity in their gene expression signal. Genotype on the horizontal axis, mean-centered expression levels on the vertical axis. (a) Polymorphism rs11741861 shows a significant association with ZNF300P1 mRNA levels in all the intestine sections, but not in the blood; (b) polymorphism rs17221417 shows different effects on NOD2 in different tissues; (c) polymorphism rs2188962 controls SLC22A5 expression uniformly and with a similar effect across different tissues.