Genome-wide association analysis of eosinophilic esophagitis provides insight into the tissue specificity of this allergic disease

Abstract

Eosinophilic esophagitis (EoE) is a chronic inflammatory disorder associated with allergic hypersensitivity to food. We interrogated >1.5 million genetic variants in EoE cases of European ancestry and subsequently in a multi-site cohort with local and out-of-study control subjects. In addition to replicating association of the 5q22 locus (meta-analysis P=1.9×10(-16)), we identified an association at 2p23 spanning CAPN14 (P=2.5×10(-10)). CAPN14 was specifically expressed in the esophagus, was dynamically upregulated as a function of disease activity and genetic haplotype and after exposure of epithelial cells to interleukin (IL)-13, and was located in an epigenetic hotspot modified by IL-13. Genes neighboring the top 208 EoE-associated sequence variants were enriched for esophageal expression, and multiple loci for allergic sensitization were associated with EoE susceptibility (4.8×10(-2)<P<5.1×10(-11)). We propose a model to explain the tissue-specific nature of EoE that involves the interplay of allergic sensitization with an EoE-specific, IL-13-inducible esophageal response involving CAPN14.

Figures

Figure 1. Manhattan plot of the p values obtained from the genome-wide association analysis

a. Data are from 736 subjects with EoE and 9,246 controls having 1,468,075 genetic variants, with minor allele frequencies greater than 1% in the subjects with EoE. The −log of the probability is shown as a function of the genomic position of the autosomes. Genome-wide significance (red dotted line, p ≤ 5×10−8) and suggestive significance (solid blue line, p ≤ 10−7) are indicated. b–e. Genetic association of variants at the 2p23, 5q22, 8p23, and 15q13 loci with EoE risk. P values (−log10) of the genetic association analysis of genotyped and imputed variants are plotted against the genomic positions of each genotyped (blue) and imputed (red) SNPs on the x axis on chromosomes 2, 5, 8, and 15. Genes in the region are shown above. The black lines indicate the recombination rates in cM per Mb using subjects of European Ancestry from the 1,000 genomes project.

Figure 2. CAPN14 is specifically expressed in esophageal epithelium, dynamically upregulated as a function of disease activity and genetic haplotype and after exposure of epithelial cells to IL-13

a. Barcode Z-score relative microarray expression of CAPN14 in various human tissue samples. Barcode analysis of 18,656 publically available microarrays as displayed by biogps.org,. Representative data from multiple cellular subtypes. Error bars represent the median absolute deviation. (Refer to Supplemental Figure 2 for an exhaustive list). b. Microarray expression heat map of calpain family in esophageal biopsies from normal controls (NL, n = 14), therapy-responsive EoE patients (EoE remission, n = 18), active EoE patients (EoE active, n = 18), and therapy-non-responsive EoE patients (EoE resistant, n = 19). c. Microarray expression analysis of CAPN14 expression from esophageal biopsies (NL, n = 14; EoE active, n = 18; EoE inactive, n = 18) (b. and c.) and primary esophageal epithelial cells with or without IL-13 stimulation for 48 hours (d). Error bars represent standard error of the mean (s.e.m.), n=3. e. Real-time PCR analysis of CAPN14 expression in biopsies from EoE patients with the non-risk haplotype (n = 19) or with at least one copy of the risk haplotype at the 2p23 loci (n = 17). The risk haplotype is defined as having the EoE risk allele (highlighted in red) at each of the six most highly associated variant locations. Error bars represent s.e.m. f. Schematic of esophageal epithelial air-liquid interface (ALI) transwell culture system and H&E staining demonstrating stratification. g. RNA-seq expression analysis of CAPN14 expression from ALI cultures with or without IL-13 stimulation for 6 days (n = 3 for each group). Error bars represent s.e.m. h. Chip-seq on TE-7 cells shows increased H3K27Ac marks with IL-13 stimulation over the transcriptional start site of CAPN14, which correlates with an increase in transcriptional activity by RNA-seq. One significantly associated SNP (rs76562819) is in this acetylation region. i. EMSA was used to probe nuclear lysates from an esophageal epithelial cell line using oligonucleotides with the risk [G] or non-risk [A] allele of rs7656219.

Figure 3. IL-13 induces CAPN14 expression and calpain activity

a. Heat map of microarray expression of the calpain family in primary esophageal epithelial cell culture (left) and in EPC2 air-liquid interface cultures (right). b. Calpain activity assay in EPC2 cells with or without IL-13 stimulation for 48 hours in the presence or absence of the calpain inhibitor PD150606. Error bars represent s.e.m. Error bars represent s.e.m. (data representative of 3 independent experiments)

Figure 4. Genes at EoE risk loci with differential expression in biopsies from EoE patients compared to controls

Genes within 25 kb of the 768 genetic variants associated with EoE (combined p −4) were used in this analysis of RNA-seq data. The expression of 208 genes was assessed. Normalized fold-change is shown for all genes with 2-fold average change and corrected p < 0.05. NL, normal controls.