High-throughput identification of regulatory elements and functional assays to uncover susceptibility genes for nasopharyngeal carcinoma

Abstract

<p>Large-scale genetic association studies have identified multiple susceptibility loci for nasopharyngeal carcinoma (NPC), but the underlying biological mechanisms remain to be explored. To gain insights into the genetic etiology of NPC, we conducted a follow-up study encompassing 6,907 cases and 10,472 controls and identified two additional NPC susceptibility loci, 9q22.33 (rs1867277; OR = 0.74, 95% CI = 0.68–0.81, p = 3.08 × 10⁻¹¹) and 17q12 (rs226241; OR = 1.42, 95% CI = 1.26–1.60, p = 1.62 × 10⁻⁸). The two additional loci, together with two previously reported genome-wide significant loci, 5p15.33 and 9p21.3, were investigated by high-throughput sequencing for chromatin accessibility, histone modification, and promoter capture Hi-C (PCHi-C) profiling. Using luciferase reporter assays and CRISPR interference (CRISPRi) to validate the functional profiling, we identified <em>PHF2</em> at locus 9q22.33 as a susceptibility gene. <em>PHF2</em> encodes a histone demethylase and acts as a tumor suppressor. The risk alleles of the functional SNPs reduced the expression of the target gene <em>PHF2</em> by inhibiting the enhancer activity of its long-range (4.3 Mb) <em>cis</em>-regulatory element, which promoted proliferation of NPC cells. In addition, we identified <em>CDKN2B-AS1</em> as a susceptibility gene at locus 9p21.3, and the NPC risk allele of the functional SNP rs2069418 promoted the expression of <em>CDKN2B-AS1</em> by increasing its enhancer activity. The overexpression of <em>CDKN2B-AS1</em> facilitated proliferation of NPC cells. In summary, we identified functional SNPs and NPC susceptibility genes, which provides additional explanations for the genetic association signals and helps to uncover the underlying genetic etiology of NPC development.</p>