Using the previously identified risk allele as an anchor, Musunuru et al. first explored whether there was a relationship between the risk variant and the expression of any of the genes on chromosome 1p13. This approach was based on the hypothesis that the risk variant might alter the abundance of a protein that is critical to the pathogenic mechanism (i.e., a bridgehead between risk allele and disease phenotype). Indeed, the investigators showed that specifically in the liver, an organ intimately involved in lipid metabolism, three genes at 1p13 were expressed at reduced levels in carriers of the risk allele.
Modulation of multiple genes by the risk allele suggested involvement of a regulatory element. In order to find this element, the investigators sequenced the 1p13 region. They found additional DNA variants and subsequently tested these variants for association with LDL cholesterol, an approach called fine mapping. They thereby narrowed down the region containing the probable causal variant to a small segment. Bioinformatic analysis indicated that one of the variants (rs12740374) within this noncoding segment lies within a putative binding site for CCAAT/enhancer binding protein (C/EBP), a transcription factor; the risk allele is predicted to disrupt binding. It was thus tempting to speculate that this particular single-nucleotide polymorphism (SNP) is directly involved in the regulation of gene expression at the chromosome 1p13 locus. Indeed, the investigators confirmed physical binding of C/EBP to a small stretch of DNA containing the protective variant but not the risk variant of rs12740374. They then observed increased expression of a reporter gene in liver cells transfected with a vector carrying the intact but not the variant C/EBP binding site. The experiments suggest a chain of events linking specifically the risk variant of SNP rs12740374 to reduced expression levels of genes on chromosome 1p13 (Figure 1).
Sortilin 1 and the Risk of Coronary Disease
The next question was how increased expression of sortilin 1 functionally results in reduced serum levels of LDL cholesterol. Sortilin 1 is localized in the Golgi apparatus and endoplasmatic reticulum, where it appears to act as an intracellular sorting receptor for particles containing apolipoprotein B.4 Musunuru et al. found that hepatocytes in mice with sortilin 1 knockdown had a significant increase in the secretion of particles containing apolipoprotein B, notably very-low-density lipoprotein (VLDL) cholesterol, whereas the overexpression of sortilin 1 was associated with decreased secretion of such particles (Figure 1). Moreover, the overexpression of sortilin 1 was shown to enhance the endocytosis of LDL particles in vitro.5 Taken together, these findings provide strong experimental evidence for the involvement of sortilin 1 in the hepatic metabolism of lipoproteins containing apolipoprotein B, although the precise mechanism awaits further elucidation.3-5
The occurrence of the risk variant of rs12740374 in multiple populations of different ancestry and its high prevalence (68% in those of European ancestry) imply that this SNP affects lipoprotein levels around the globe through its modulatory effects on gene transcription.3 The resulting decrease of sortilin 1 expression appears to elevate levels of two atherogenic lipoproteins, VLDL and LDL, and thus acts as a double-edged sword with respect to atherosclerosis risk. The clinical importance of this pathway is defined by a substantial increase in the risk of coronary disease for every copy of the 1p13 risk allele.1,2,5 All told, the discovery of the C/EBP–sortilin 1 pathway provides an example of how genomewide association studies can initiate new biologic discoveries, leading to a better understanding of pathophysiology, which could ultimately translate into new approaches to treatment.
