Whole-exome sequencing identifies rare and low-frequency coding variants associated with LDL cholesterol

Abstract

Elevated low-density lipoprotein cholesterol (LDL-C) is a treatable, heritable risk factor for cardiovascular disease. Genome-wide association studies (GWASs) have identified 157 variants associated with lipid levels but are not well suited to assess the impact of rare and low-frequency variants. To determine whether rare or low-frequency coding variants are associated with LDL-C, we exome sequenced 2,005 individuals, including 554 individuals selected for extreme LDL-C (>98(th) or <2(nd) percentile). Follow-up analyses included sequencing of 1,302 additional individuals and genotype-based analysis of 52,221 individuals. We observed significant evidence of association between LDL-C and the burden of rare or low-frequency variants in PNPLA5, encoding a phospholipase-domain-containing protein, and both known and previously unidentified variants in PCSK9, LDLR and APOB, three known lipid-related genes. The effect sizes for the burden of rare variants for each associated gene were substantially higher than those observed for individual SNPs identified from GWASs. We replicated the PNPLA5 signal in an independent large-scale sequencing study of 2,084 individuals. In conclusion, this large whole-exome-sequencing study for LDL-C identified a gene not known to be implicated in LDL-C and provides unique insight into the design and analysis of similar experiments.

Figure 1

Effect Sizes Observed for Gene-Based Burden Tests Relative to GWAS Variants Effect sizes are shown in SD units for genes identified by burden tests of nonsynonymous (NS) and splice variants (blue), LoF variants only (red), and GWAS variants (black) from Willer et al. For genes, the burden effect sizes and burden frequencies are plotted. For GWAS variants, the observed effect sizes and MAFs are plotted. The burden frequencies for the gene-based tests (red and black) were observed in this study, whereas the GWAS-variant frequencies are plotted as reported in Willer et al. The alpha level was set to 5 × 10⁻⁷ to reflect the significance threshold used for gene-based burden tests.

Figure 2

LDL-C Values for Individuals with Different Types of Genetic Variants in Four LDL-C-Associated Genes A representation of LDL-C values for each individual with a rare variant in (A) PCSK9, (B) LDLR, (C) APOB, or (D) PNPLA5. The left side of the figure shows LDL-C levels per individual with a rare allele in the gene. On the right, bean plots indicate the mean (black line) and distribution (bean shape) of LDL-C values for individuals with a LoF variant, a missense variant, or no rare variant. (A) Individuals classified on the basis of variants in PCSK9 (MAF < 5% is considered rare). (B) Individuals classified by carrier status of variants in LDLR (MAF < 0.1% is considered rare on the basis of the most significant burden test for this gene). (C) Individuals classified by genetic status at APOB (MAF < 5% is considered rare). (D) Individuals with a rare variant in PNPLA5 (nonsynonymous or splice with MAF < 0.1%) are shown.