Trans-ancestry Fine Mapping and Molecular Assays Identify Regulatory Variants at the ANGPTL8 HDL-C GWAS Locus

Figure 1. Thirteen variants overlap predicted regulatory regions. Thirteen variants overlap regulatory regions defined by histone marks, chromatin accessibility, and transcription factor binding. The full set of 42 candidate variants span a 39-kb region (Figure S4 in File S1). Green rectangles denote DNase hypersensitivity peaks correlated with ANGPTL8 expression across 112 cell lines in a previous study (Sheffield et al. 2013). DNase hypersensitivity correlation with DOCK6 expression is not indicated because the correlated peaks do not overlap HepG2 or hepatocyte DNase peaks. Gray rectangles represent transcription factor binding; the identities of transcription factors are listed in Table S5 in File S1. Data were accessed from ENCODE, the Epigenome Roadmap Atlas, and the UCSC Genome Browser. ENCODE ChromHMM: gray, heterochromatin; blue, insulator; green, transcription; yellow and orange, enhancer; red and pink, promoter. Roadmap ChromHMM: orange, enhancer; light green, genic enhancer; dark green, transcription; blue, heterochromatin; red, promoter. Black rectangles represent regions analyzed in transcriptional reporter assays.

Figure 2. Cell-type specificity of ANGPTL8 is influenced by nearby regulatory regions. Candidate regulatory regions were tested in a pGL4.10 vector in six cell types to determine drivers of tissue specificity. Reporter activity was normalized to empty vector (EV) in each cell type. Data are represented as the mean ± SD of 10 biological replicates. (A) A 400-bp promoter. (B) A 5-kb regulatory region including the promoter. Comparison of the 400-bp promoter vs. 5-kb regulatory region; P < 0.05 for cell types. Cell lines: HepG2, human hepatocellular carcinoma (liver); Huh-7, human hepatocellular carcinoma; SGBS, human preadipocyte; SW872, human adipocyte; 3T3L1, mouse adipocyte; MIN6, mouse pancreatic β cell; 293T, human embryonic kidney.

Figure 3. Locus plots of HDL-C and eQTL associations near ANGPTL8. (A) Concentration of phospholipids in medium HDL in the METSIM study of Finnish individuals (N = 8380). Variants are colored according to LD (r2) with rs737337 (purple), the lead variant in European meta-analyses by GLGC. (B) HDL-C association in the WHI subset of African American individuals (N = 8244). Variants are colored according to LD (r2) with rs4804154 (purple). (C) HDL-C–associated variants are also associated with ANGPTL8 expression in 770 subcutaneous adipose samples from the METSIM study. The European and African American lead variants (rs737337 and rs4804154, labeled in plots) are among the most significant variants. Variants are colored according to LD (r2) with rs4804154 (purple).

Figure 4. Allelic differences in regulatory assays of rs12463177 (A) A 698-bp region containing either allele of rs12463177 was cloned into the pGL4.23 vector and transfected in HepG2 cells. Data are represented as the mean ± SD of 3–5 biological replicates. Luciferase activity was normalized to empty vector (EV) and P-values were determined by t-tests. * P < 0.05, ** P < 0.01. (B) Differential protein binding was evaluated in vitro using EMSA. IR-labeled probes containing either allele of rs12463177 were incubated with 10 μg HepG2 nuclear protein. The arrow shows stronger binding for rs12463177-G. Consistent binding was observed with SGBS nuclear protein (Figure S7 in File S1). HDL-C–increasing alleles are presented first.

Figure 5. Summary of functional results for seven candidate variants. Summary of shared LD with lead HDL-C–associated variants reported in European (rs737337), African American (rs4804154), and East Asian (rs3760782) populations are shown; variants meeting the r2 > 0.5 threshold are marked with +. Results from transcriptional reporter luciferase assays and electrophoretic mobility shift assays (EMSA) for nine candidate variants are shown. Arrows show the direction of transcriptional activity in reporter assays. Two arrows at rs12463177-G indicate allelic differences in transcriptional activity. Black circles show allele-specific protein binding observed in EMSA experiments. The larger and smaller arrows at the ANGPTL8 promoter indicate higher and lower expression level from adipose eQTL data. Variants are located within 9 kb of the ANGPTL8 transcription start site; variant distances are not drawn to scale.