Genetic susceptibility to Barrett's esophagus: From GWAS to biology

Barrett’s esophagus (BE) is the only known precursor for esophageal adenocarcinoma (EAC), a highly lethal
cancer with rising incidence and median survival <1 year. Substantial health-care resources are devoted to BE
screening, surveillance, and treatment. Gastroesophageal reflux-induced injury of the lower esophagus and
chronic inflammation are key drivers of BE development, but molecular pathways underlying risk are not well
defined. Recent genome-wide association studies (GWAS) led by members of our team identified >20 novel
genetic susceptibility loci for BE/EAC, providing new insights into the inherited genetic component of risk.
Nevertheless, little progress has been made in bridging associations to biology. Consistent with GWAS of other
complex diseases, all BE index variants map to non-coding regions, lack obvious biologic function, and are in
linkage disequlibrium with many other SNPs, any of which may be causal. The vast majority of functional
variants underlying GWAS signals are believed to map to and alter activity of regulatory elements including
enhancers, in an allele-specific manner, and in turn modulate expression of downstream genes involved in risk.
Importantly, such regulatory effects may be tissue- and condition-specific. To begin prioritizing candidate
functional variants for experimental interrogation, we developed a customized informatics scoring pipeline
using comprehensive in-silico annotations from multiple public resources. We selected four high-scoring BE
risk loci for evaluation using luciferase reporter assays in esophageal cell lines, and found that two of four
regions exhibited allele-specific enhancer activity. CRISPR-mediated deletion of the enhancer region at both
loci correlated with downregulation of several candidate risk genes. Motivated by these successes, we seek to
expand our integrative framework for elucidating functional consequences of BE-related genetic variation. We
hypothesize that such variation is biologically expressed through alterations in transcriptional regulation and
downstream gene expression. Our goal is to identify functional variants, risk enhancers, and target genes
underlying BE risk, leveraging unique resources and complementary statistical/experimental approaches. In
Aim 1, we will define candidate causal variants via Bayesian fine-mapping, using the largest BE GWAS world-
wide, and further prioritize leading candidates via functional-potential scores. In Aim 2, we will perform new
transcriptome profiling of reflux-exposed gastroesophageal junction tissues and constituent cells, and identify
candidate BE risk genes and pathways via eQTL colocalization and network-based analysis. In Aim 3, we will
validate candidate functional variants using luciferase reporter enhancer activity assays; identify target genes
of risk enhancers via CRISPR-mediated enhancer deletion and RNA-Seq; and interrogate pathways influenced
by prioritized target genes in Aims 2 & 3 via CRISPR-mediated gene knockdown/overexpression and RNA-
Seq. This study will advance noncoding GWAS signals into functional biological signatures and support future
efforts to develop novel preventive/interventional strategies for BE/EAC.

Grant Number: 5R01DK128615-04
NIH Institute/Center: NIH
Principal Investigator: Matthew Buas