title: Identification of the Functional Variant(s) that Explain the Low-Density Lipoprotein Receptor (LDLR) GWAS SNP rs6511720 Association with Lower LDL-C and Risk of CHD.
abstract: BACKGROUND
  The Low-Density Lipoprotein Receptor (LDLR) SNP rs6511720 (G>T), located in intron-1 of the gene, has been identified in genome-wide association studies (GWAS) as being associated with lower plasma levels of LDL-C and a lower risk of coronary heart disease (CHD). Whether or not rs6511720 is itself functional or a marker for a functional variant elsewhere in the gene is not known.
  
  
  METHODS
  The association of LDLR SNP rs6511720 with incidence of CHD and levels of LDL-C was determined by reference to CARDIoGRAM, C4D and Global lipids genetics consortium (GLGC) data. SNP annotation databases were used to identify possible SNP function and prioritization. Luciferase reporter assays in the liver cell line Huh7 were used to measure the effect of variant genotype on gene expression. Electrophoretic Mobility Shift Assays (EMSAs) were used to identify the Transcription Factors (TFs) involved in gene expression regulation.
  
  
  RESULTS
  The phenotype-genotype analysis showed that the rs6511720 minor allele is associated with lower level of LDL-C [beta = -0.2209, p = 3.85 x10-262], and lower risk of CHD [log (OR) = 0.1155, p = 1.04 x10-7]. Rs6511720 is in complete linkage. Rs6511720 is in complete linkage disequilibrium (LD) with three intron-1 SNPs (rs141787760, rs60173709, rs57217136). Luciferase reporter assays in Huh7 cells showed that the rare alleles of both rs6511720 and rs57217136 caused a significant increase in LDLR expression compared to the common alleles (+29% and +24%, respectively). Multiplex Competitor-EMSAs (MC-EMSA) identified that the transcription factor serum response element (SRE) binds to rs6511720, while retinoic acid receptor (RAR) and signal transducer and activator of transcription 1 (STAT1) bind to rs57217136.
  
  
  CONCLUSIONS
  Both LDLR rs6511720 and rs57217136 are functional variants. Both these minor alleles create enhancer-binding protein sites for TFs and may contribute to increased LDLR expression, which is consequently associated with reduced LDL-C levels and 12% lower CHD risk.
authors.affiliation: Centre for Cardiovascular Genetics, BHF Laboratories, Institute of Cardiovascular Science, University College London, London, United Kingdom.
authors.forename: Roaa Hani
authors.initials: RH
authors.lastname: Fairoozy
authors.affiliation: University College London Genetics Institute, Department of Genetics, Environment and Evolution, London, United Kingdom.
authors.forename: Jon
authors.initials: J
authors.lastname: White
authors.affiliation: Centre for Cardiovascular Genetics, BHF Laboratories, Institute of Cardiovascular Science, University College London, London, United Kingdom.
authors.forename: Jutta
authors.initials: J
authors.lastname: Palmen
authors.affiliation: Centre for Cardiovascular Genetics, BHF Laboratories, Institute of Cardiovascular Science, University College London, London, United Kingdom.
authors.forename: Anastasia Z
authors.initials: AZ
authors.lastname: Kalea
authors.affiliation: Centre for Cardiovascular Genetics, BHF Laboratories, Institute of Cardiovascular Science, University College London, London, United Kingdom.
authors.forename: Steve E
authors.initials: SE
authors.lastname: Humphries
mesh_terms: D002460:Cell Line; D008078:Cholesterol, LDL / Q000378:metabolism*; D003324:Coronary Artery Disease / Q000235:genetics*; D024202:Electrophoretic Mobility Shift Assay; D020022:Genetic Predisposition to Disease / Q000235:genetics; D055106:Genome-Wide Association Study; D005838:Genotype; D006801:Humans; D007438:Introns / Q000235:genetics; D015810:Linkage Disequilibrium / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D011973:Receptors, LDL / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D008078:Cholesterol, LDL; C573412:LDLR protein, human; D011973:Receptors, LDL; D014157:Transcription Factors