Systematic identification of genotype-dependent enhancer variants in eosinophilic esophagitis and atopic dermatitis [MPRA]
Eosinophilic esophagitis (EoE) is a rare atopic disorder associated with esophageal dysfunction, including difficulty swallowing, food impaction, and inflammation.  EoE develops in a small subset of people with food allergies under the influence of environmental and genetic risk factors.  Genome wide association studies (GWAS) have identified 31 independent risk loci for the disease, and linkage disequilibrium (LD) expansion of these loci nominates a set of 531 variants that are potentially causal.  These risk variants are non-coding, suggesting a likely role in altered gene regulatory mechanisms. To systematically interrogate the gene regulatory activity of these variants, we designed a massively parallel reporter assay (MPRA) containing the alleles of each variant within their 170 bp genomic sequence context cloned into a GFP reporter library.  Transfection of the MPRA library into TE-7 esophageal epithelial cells, HaCaT skin keratinocytes, and Jurkat T cells revealed cell-type-specific gene regulation.  We identify 32 allelic enhancer variants (allelic enVars) that regulate reporter gene expression in a genotype-dependent manner in at least one cellular context.  By annotating these variants with expression quantitative trait loci (eQTL) and chromatin looping data in related tissues and cell types, we identify putative target genes affected by genetic variation in EoE patients, including TSLP and multiple genes at the HLA locus. Transcription factor enrichment analyses reveal possible roles for cell-type specific regulators, including GATA-3, a key regulator of type 2 inflammation. Collectively, our approach reduces the large set of EoE-associated variants to a set of 32 with allelic regulatory activity, providing new functional insights into the effects of genetic variation in this disease. Overall design: Massively parallel reporter assays to study EoE and atopic dermatitis-associated variants for allele-dependent transcriptional enhancer activity, 5 replicates, 2 conditions, 3 cell types