Mining the LIPG allelic spectrum reveals the contribution of rare and common regulatory variants to HDL cholesterol

Abstract

Genome-wide association studies (GWAS) have successfully identified loci associated with quantitative traits, such as blood lipids. Deep resequencing studies are being utilized to catalogue the allelic spectrum at GWAS loci. The goal of these studies is to identify causative variants and missing heritability, including heritability due to low frequency and rare alleles with large phenotypic impact. Whereas rare variant efforts have primarily focused on nonsynonymous coding variants, we hypothesized that noncoding variants in these loci are also functionally important. Using the HDL-C gene LIPG as an example, we explored the effect of regulatory variants identified through resequencing of subjects at HDL-C extremes on gene expression, protein levels, and phenotype. Resequencing a portion of the LIPG promoter and 5' UTR in human subjects with extreme HDL-C, we identified several rare variants in individuals from both extremes. Luciferase reporter assays were used to measure the effect of these rare variants on LIPG expression. Variants conferring opposing effects on gene expression were enriched in opposite extremes of the phenotypic distribution. Minor alleles of a common regulatory haplotype and noncoding GWAS SNPs were associated with reduced plasma levels of the LIPG gene product endothelial lipase (EL), consistent with its role in HDL-C catabolism. Additionally, we found that a common nonfunctional coding variant associated with HDL-C (rs2000813) is in linkage disequilibrium with a 5' UTR variant (rs34474737) that decreases LIPG promoter activity. We attribute the gene regulatory role of rs34474737 to the observed association of the coding variant with plasma EL levels and HDL-C. Taken together, the findings show that both rare and common noncoding regulatory variants are important contributors to the allelic spectrum in complex trait loci.

Figure 1. Rare and common LIPG regulatory variants studied.

Diagram of Chr. 18q21.1 region containing LIPG with variants identified annotated.

Figure 2. Rare LIPG regulatory variants modulate transcriptional activity in vitro.

Relative promoter activity of rare variants (MAF<0.01) identified from resequencing of high HDL-C individuals (A) or low HDL-C individuals (B). Plasmid constructs expressing firefly luciferase under the control of wild-type (WT) or variant LIPG promoters were individually co-transfected with a Renilla luciferase reporter construct (pRL-SV40) in HUVECs. Firefly luciferase expression were measured and normalized to that of Renilla luciferase, and Renilla-normalized promoter activities for variant constructs were then compared to those of the WT construct to provide relative LIPG promoter activities of the variants. Assays were conducted with 6 replicates per experiment and data is given as mean ± standard deviation. *P-value<0.05, **P-value<0.01, ***P-value<0.0001, compared with WT.

Figure 3. Common LIPG regulatory variant rs34474737 affects LIPG promoter activity in vitro.

Relative LIPG promoter activity of common variants rs34474737 (229 T>G) and −1358 T insertion variant identified from resequencing of individuals with high and low HDL-C levels, measured as relative firefly luciferase expression of LIPG variant constructs in HUVECs. Assays were conducted with 6 replicates per experiment and data is given as mean ± standard deviation. **P-value<0.01, compared with WT.

Figure 4. Linkage disequilibrium of rs34474737 (229 T>G) and rs2000813 (Thr111Ile) variants.

Genotyping of rs34474737 and rs2000813 variants was completed in SIRCA participants (761 in total). LD was estimated and plotted using this genotyping data using Haploview software. Values in the LD plot are estimated squared correlation coefficients (R²).