Identification of the Functional Variant(s) that Explain the Low-Density Lipoprotein Receptor (<i>LDLR</i>) GWAS SNP rs6511720 Association with Lower LDL-C and Risk of CHD

<div><p>Background</p><p>The Low-Density Lipoprotein Receptor (<i>LDLR</i>) 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.</p><p>Methods</p><p>The association of <i>LDLR</i> 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.</p><p>Results</p><p>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 <i>LDLR</i> 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.</p><p>Conclusion</p><p>Both <i>LDLR</i> rs6511720 and rs57217136 are functional variants. Both these minor alleles create enhancer-binding protein sites for TFs and may contribute to increased <i>LDLR</i> expression, which is consequently associated with reduced LDL-C levels and 12% lower CHD risk.</p></div

DNA binding properties of <i>LDLR</i> intron-1 SNPs.

<p>Conventional EMSA analysis of the <i>LDLR</i> intron-1 SNPs (rs6511720, rs141687760, rs60173709, and rs57217136). Binding of SREBP1 was used as the control (lane 1 and 2). The lanes with a labeled probe showed a specific band indicated by arrows, while when the unlabeled probe was added the band disappeared. These four SNPs have allele-specific binding, indicated by arrows. (-) = deletion and (*) = minor allele.</p

<i>LDLR</i> luciferase constructs and SNP luciferase activity in Huh7 cell line.

<p>A) Schematic presentation of LDLR<b>-</b>luciferase-construct (promoter only) and LDLR<b>-</b>luciferase-enhancer-constructs. The constructs were transfected into Huh7 cells. B) Results of luciferase reporter assays showing relative expression of LDLR-luciferase-enhancer constructs of <i>LDLR</i> SNPs relative to the LDLR-luciferase (no enhancer) construct. (-) = deletion and (*) = minor allele.</p

Genome-wide maps of chromatin state of LDLR intron-1.

<p>Schematic presentation of the LDLR intron-1 chromatin status (<a href="https://genome-euro.ucsc.edu" target="_blank">https://genome-euro.ucsc.edu</a>). The area of interest in intron-1 is highlighted in light-blue color. Promoter/ Enhancer histone marker of seven cell lines (GM12878, H1-hESC, HSMM, HUVEC, K562, NHEK, and NHLF). FAIRE: formaldehyde assisted isolation of regulatory elements.</p

Meta-analysis association results for hemoglobin in unconditional and conditional analyses in the <i>ABO</i> locus.

<p>Regional plots show (A) unconditional analysis and analysis conditional on (B) O, (C) AO, (D) AA, (E) B and (F) AB blood group haplotype. The most significant SNP in the unconditional analysis, rs507666, is highlighted throughout to facilitate comparison of results. The color coding of the LD between the SNPs ranges from dark blue for r² = 0–0.2 to red for r² = 0.8–1, and is grey where LD information was not available. Blue line represents suggestive and red line significant threshold.</p

Comparison of the effect sizes on 15 <i>ABO</i> SNPs between eight different traits.

<p>Traits include von Willebrand factor (log transformed, logVWF) and factor VIII (FVIII) for coagulation factors, total cholesterol (TC) and low-density lipoprotein (LDL) for lipids, hemoglobin (Hb), red blood cell count (RCC) and hematocrit (Hct) for the red blood cell (RBC) traits and alkaline phosphatase (log transformed, logALP) for liver marker group. The colored bar for each SNP represents the 95% confidence interval of the effect size.</p

Study characteristics.

<p>Study characteristics.</p

Results of meta-analysis and replication analysis for 15 most significant SNPs in the <i>ABO</i> locus.

<p>Results of meta-analysis and replication analysis for 15 most significant SNPs in the <i>ABO</i> locus.</p

Blood groups derived based on three SNP haplotypes.

<p>Blood groups derived based on three SNP haplotypes.</p

Manhattan plots of meta-analysis association results in unconditional and conditional analyses for hemoglobin (Hb), hematocrit (Hct) and red blood cell count (RCC).

<p>Results show unconditional analysis for all three traits (top row) and analysis of Hb conditional on (A) Hct and (B) RCC, of Hct conditional on (C) Hb and (D) RCC and of RCC conditional on (E) Hb and (F) Hct. Line at–log₁₀(<i>P</i> value) = 5.3 represents suggestive threshold and line at–log₁₀(<i>P</i> value) = 7.3 significant threshold.</p