A risk variant for Barrett's esophagus and esophageal adenocarcinoma at chr8p23.1 affects enhancer activity and implicates multiple gene targets

Abstract

Nineteen genetic susceptibility loci for esophageal adenocarcinoma (EAC) and its precursor Barrett's esophagus (BE) have been identified through genome-wide association studies (GWAS). Clinical translation of such discoveries, however, has been hindered by the slow pace of discovery of functional/causal variants and gene targets at these loci. We previously developed a systematic informatics pipeline to prioritize candidate functional variants using functional potential scores, applied the pipeline to select high-scoring BE/EAC risk loci and validated a functional variant at chr19p13.11 (rs10423674). Here, we selected two additional prioritized loci for experimental interrogation: chr3p13/rs1522552 and chr8p23.1/rs55896564. Candidate enhancer regions encompassing these variants were evaluated using luciferase reporter assays in two EAC cell lines. One of the two regions tested exhibited allele-specific enhancer activity - 8p23.1/rs55896564. CRISPR-mediated deletion of the putative enhancer in EAC cell lines correlated with reduced expression of three candidate gene targets: B lymphocyte kinase (BLK), nei like DNA glycosylase 2 (NEIL2) and cathepsin B (CTSB). Expression quantitative trait locus (eQTL) mapping in normal esophagus and stomach revealed strong associations between the BE/EAC risk allele at rs55896564 (G) and lower expression of CTSB, a protease gene implicated in epithelial wound repair. These results further support the utility of functional potential scores for GWAS variant prioritization, and provide the first experimental evidence of a functional variant and risk enhancer at the 8p23.1 GWAS locus. Identification of CTSB, BLK and NEIL2 as candidate gene targets suggests that altered expression of these genes may underlie the genetic risk association at 8p23.1 with BE/EAC.

Figure 1

Genome Browser plots for selected GWAS risk loci. (A) chr3p13/FOXP1, (B) chr8p23.1/LINC00208/BLK). AVS, associated variant set; red: index variants, blue r² > 0.80. Enh, predicted enhancer regions based on H3-K27ac ChIP-seq profiles in specific primary tissues: gastric (dark green), stomach smooth muscle (light green) (Hsniz 2013). Candidate enhancer regions, CER; CER1: 3:70906815-70 907 701 (rs1522552); CER2: 8:11446898-11 447 336 (rs17153498/rs55896564/56051089) [http://genome.ucsc.edu]

Figure 2

Luciferase reporter enhancer activity assays. The candidate enhancer region at chr8p23.1 (CER2/rs17153498/rs55896564/rs56051089) was assessed for allele-specific activity, alongside positive and negative control fragments. Three independent experiments were performed in triplicate in two EAC cell lines: (A) OE19 and (B) OE33.

Figure 3

Assessment of protein binding and DNA sequence motifs at CER2/rs55896564. (A) Human transcription factor ChIP-seq profiles in upper-GI-tract tissues and EAC cell lines. Two proteins showed evidence of local binding: KLF5 (ESO-26 cell line) and CTCF (esophagus, stomach, GE junction). Peak maximum indicated by vertical bar in binding region [http://genome.ucsc.edu]. (B) Predicted impact of rs55896564 on motif scores of overlapping KLF DNA-binding motifs identified via motifbreakR; motif scores computed using input sequences bearing the A or G allele are listed [pct_A, pct_G]. (C) Motif logo for KLF4; the position of rs55896564 indicated by dotted box.

Figure 4

CRISPR-Cas9 genome editing of putative enhancer at chr8p23.1. DNA gel electrophoresis showing genome editing of the chr8p23.1 candidate enhancer region containing variants rs17153498, rs55896564 and rs56051089. 1.5 kb band demonstrates targeted deletion of the putative enhancer in OE19 and OE33 cells. gRNA (−): cells electroporated with mock conditions; gRNA (+): cells electroporated with cas9 vector and guide RNA target vectors.

Figure 5

Gene expression changes following CRISPR-Cas9 deletion of putative enhancer at 8p23.1. (A) UCSC Genome Browser plot of ~500 kb genomic region centered on CER2 (highlighted). (B) CER2 was targeted for deletion in OE19 and OE33 cells using CRISPR-Cas9 technology. Pools of transfected cells were analyzed using TaqMan gene expression assays for BLK, GATA4, NEIL2 and CTSB, in triplicate, in three independent experiments. NC: mock electroporated parental cells; Cas9 + gRNA: cells electroporated with Cas9 vector and guide RNA target vectors. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; and ^****P < 0.0001.