title: The type 2 diabetes presumed causal variant within TCF7L2 resides in an element that controls the expression of ACSL5.
abstract: 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.
authors.affiliation: Divisions of Human Genetics and Endocrinology, The Children's Hospital of Philadelphia, 3615 Civic Center Boulevard, Room 1102D, Philadelphia, PA, 19104, USA.
authors.forename: Qianghua
authors.initials: Q
authors.lastname: Xia
authors.affiliation: Divisions of Human Genetics and Endocrinology, The Children's Hospital of Philadelphia, 3615 Civic Center Boulevard, Room 1102D, Philadelphia, PA, 19104, USA.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Chesi
authors.affiliation: Divisions of Human Genetics and Endocrinology, The Children's Hospital of Philadelphia, 3615 Civic Center Boulevard, Room 1102D, Philadelphia, PA, 19104, USA.|Institute for Biomedical Informatics, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Manduchi
authors.affiliation: Divisions of Human Genetics and Endocrinology, The Children's Hospital of Philadelphia, 3615 Civic Center Boulevard, Room 1102D, Philadelphia, PA, 19104, USA.
authors.forename: Brian T
authors.initials: BT
authors.lastname: Johnston
authors.affiliation: Divisions of Human Genetics and Endocrinology, The Children's Hospital of Philadelphia, 3615 Civic Center Boulevard, Room 1102D, Philadelphia, PA, 19104, USA.
authors.forename: Sumei
authors.initials: S
authors.lastname: Lu
authors.affiliation: Divisions of Human Genetics and Endocrinology, The Children's Hospital of Philadelphia, 3615 Civic Center Boulevard, Room 1102D, Philadelphia, PA, 19104, USA.
authors.forename: Michelle E
authors.initials: ME
authors.lastname: Leonard
authors.affiliation: Divisions of Human Genetics and Endocrinology, The Children's Hospital of Philadelphia, 3615 Civic Center Boulevard, Room 1102D, Philadelphia, PA, 19104, USA.
authors.forename: Ursula W
authors.initials: UW
authors.lastname: Parlin
authors.affiliation: NAPCore, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.
authors.forename: Eric F
authors.initials: EF
authors.lastname: Rappaport
authors.affiliation: Division of Hematology, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.
authors.forename: Peng
authors.initials: P
authors.lastname: Huang
authors.affiliation: Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Wells
authors.affiliation: Division of Hematology, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.|Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: Gerd A
authors.initials: GA
authors.lastname: Blobel
authors.affiliation: Divisions of Human Genetics and Endocrinology, The Children's Hospital of Philadelphia, 3615 Civic Center Boulevard, Room 1102D, Philadelphia, PA, 19104, USA.
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Johnson
authors.affiliation: Divisions of Human Genetics and Endocrinology, The Children's Hospital of Philadelphia, 3615 Civic Center Boulevard, Room 1102D, Philadelphia, PA, 19104, USA. grants@chop.edu.|Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. grants@chop.edu.|Institute of Diabetes, Obesity and Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. grants@chop.edu.
authors.forename: Struan F A
authors.initials: SFA
authors.lastname: Grant
mesh_terms: D015153:Blotting, Western; D064130:CRISPR-Associated Proteins / Q000378:metabolism; D002460:Cell Line; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000235:genetics* / Q000378:metabolism*; D003106:Colon / Q000378:metabolism; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics* / Q000378:metabolism*; D045325:HCT116 Cells; D006801:Humans; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D034741:RNA, Small Interfering; D020133:Reverse Transcriptase Polymerase Chain Reaction; D058118:Transcription Factor 7-Like 2 Protein / Q000235:genetics* / Q000378:metabolism*
chemical_list: D064130:CRISPR-Associated Proteins; D002843:Chromatin; D034741:RNA, Small Interfering; D058118:Transcription Factor 7-Like 2 Protein; D003066:Coenzyme A Ligases; C485465:ACSL5 protein, human
keywords: ACSL5; CRISPR; Chromatin conformation capture; Expression; TCF7L2