Center for Public Health Genomics, Department of Public Health Sciences, University of Virginia, Charlottesville, VA 22903, USA.
Department of Cancer Prevention and Control, Roswell Park Comprehensive Cancer Center, Buffalo, NY 14263, USA.
Hum Mol Genet. 2022 Nov 28;31(23):3975-3986. doi: 10.1093/hmg/ddac141.
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.
通过全基因组关联研究（GWAS），已经确定了 19 个与食管腺癌（EAC）及其前体 Barrett 食管（BE）相关的遗传易感性位点。然而，这些发现的临床转化受到在这些位点发现功能/因果变异和基因靶标的缓慢速度的阻碍。我们之前开发了一种系统的信息学管道，使用功能潜力评分来优先考虑候选功能变体，应用该管道选择高评分的 BE/EAC 风险位点，并验证了 chr19p13.11（rs10423674）的功能变体。在这里，我们选择了另外两个需要实验研究的优先级位点：chr3p13/rs1522552 和 chr8p23.1/rs55896564。使用两个 EAC 细胞系中的荧光素酶报告基因检测评估包含这些变体的候选增强子区域。在两个测试的区域中，一个区域表现出等位基因特异性增强子活性 - chr8p23.1/rs55896564。在 EAC 细胞系中 CRISPR 介导的假定增强子缺失与三个候选基因靶标（B 淋巴细胞激酶（BLK）、neil 样 DNA 糖基化酶 2（NEIL2）和组织蛋白酶 B（CTSB））的表达降低相关。在正常食管和胃中的表达数量性状基因座（eQTL）图谱中，rs55896564（G）的 BE/EAC 风险等位基因与 CTSB 的表达降低之间存在强烈关联，CTSB 是一种参与上皮伤口修复的蛋白酶基因。这些结果进一步支持了功能潜力评分在 GWAS 变体优先级中的应用，并且提供了在 8p23.1 GWAS 位点的第一个功能变体和风险增强子的实验证据。鉴定 CTSB、BLK 和 NEIL2 作为候选基因靶标表明，这些基因的表达改变可能是 8p23.1 与 BE/EAC 的遗传风险关联的基础。
