The type 2 diabetes presumed causal variant within TCF7L2 resides in an element that controls the expression of ACSL5

Qianghua Xia, Alessandra Chesi, Elisabetta Manduchi, Brian T. Johnston, Sumei Lu, Michelle E. Leonard, Ursula W. Parlin, Eric F. Rappaport, Peng Huang, Andrew D. Wells, Gerd A. Blobel, Matthew E. Johnson, Struan F. A. Grant

One of the most strongly associated type 2 diabetes loci reported to date resides within the TCF7L2 gene. Previous studies point to the T allele of rs7903146 in intron 3 as the causal variant at this locus. We aimed to identify the actual gene(s) under the influence of this variant. Using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease, we generated a 1.4 kb deletion of the genomic region harbouring rs7903146 in the HCT116 cell line, followed by global gene expression analysis. We then carried out a combination of circularised chromosome conformation capture (4C) and Capture C in cell lines, HCT116 and NCM460 in order to ascertain which promoters of these perturbed genes made consistent physical contact with this genomic region. We observed 99 genes with significant differential expression (false discovery rate [FDR] cut-off:10%) and an effect size of at least twofold. The subsequent promoter contact analyses revealed just one gene, ACSL5, which resides in the same topologically associating domain as TCF7L2. The generation of additional, smaller deletions (66 bp and 104 bp) comprising rs7903146 showed consistently reduced ACSL5 mRNA levels across all three deletions of up to 30-fold, with commensurate loss of acyl-CoA synthetase long-chain family member 5 (ACSL5) protein. Notably, the deletion of this single-nucleotide polymorphism region abolished significantly detectable chromatin contacts with the ACSL5 promoter. We went on to confirm that contacts between rs7903146 and the ACSL5 promoter regions were conserved in human colon tissue. ACSL5 encodes ACSL5, an enzyme with known roles in fatty acid metabolism. This 'variant to gene mapping' effort implicates the genomic location harbouring rs7903146 as a regulatory region for ACSL5.