Genomewide Association Analysis and Relationship to Cardiovascular Risk and Mortality in Older Adults

Objective
CD14 is a glycosylphosphatidylinositol-anchored membrane glycoprotein expressed on neutrophils and monocytes/macrophages that also circulates as a soluble form (sCD14). Despite the well-recognized role of CD14 in inflammation, relatively little is known about the genetic determinants of sCD14 or the relationship of sCD14 to vascular- and aging-related phenotypes.

Methods and Results
We measured baseline levels of sCD14 in >5000 European-American and black adults aged 65 years and older from the Cardiovascular Health Study, who were well characterized at baseline for atherosclerotic risk factors and subclinical cardiovascular disease, and who have been followed for clinical cardiovascular disease and mortality outcomes up to 20 years. At baseline, sCD14 generally showed strong positive correlations with traditional cardio-metabolic risk factors and with subclinical measures of vascular disease such as carotid wall thickness and ankle-brachial index (independently of traditional cardiovascular disease risk factors), and was also inversely correlated with body mass index. In genomewide association analyses of sCD14, we (1) confirmed the importance of the CD14 locus on chromosome 5q21 in European-American; (2) identified a novel African ancestry-specific allele of CD14 associated with lower sCD14 in blacks; and (3) identified a putative novel association in European-American of a nonsynonymous variant of PIGC, which encodes an enzyme required for the first step in glycosylphosphatidylinositol anchor biosynthesis. Finally, we show that, like other acute phase inflammatory biomarkers, sCD14 predicts incident cardiovascular disease, and strongly and independently predicts all-cause mortality in older adults.

Key Words: CD14, coronary heart disease, glycosylphosphatidylinositol, mortality.

CD14 and sCD14 are able to mediate LPS-activation of mCD14-negative cells such as endothelial and smooth muscle cells,11,12 events that are important for the development of atherosclerosis and its complications. At higher concentrations, LPS binding protein and sCD14 downregulate LPS-induced responses13 by transfer of LPS to lipoproteins for subsequent removal. CD14 is a membrane glycoprotein constitutively expressed on monocytes/macrophages and neutrophils.1 Cell surface or membrane (m)CD14 binds to lipopolysaccharide (LPS) and other microbial and nonmicrobial ligands in the presence of another accessory protein, LPS binding protein, resulting in activation of various toll-like receptors and downstream pro-inflammatory pathway signaling.2,3 CD14 also exists as a soluble form (sCD14).4 In addition to secretion from liver, sCD14 may be derived by enzymatic cleavage of GPI-anchored cell membrane-bound CD14.

The genetic and environmental determinants of sCD14 are largely unknown, though CD14 expression appears to be at least partially regulated at the genetic level in a cell-type specific manner. The human CD14 gene on chromosome 5q23-31 contains a common proximal promoter polymorphism encoding a cytosine to thymidine transition at bp 159 from the major transcription start site that has been associated with circulating sCD14 levels.14,15 A regulatory element located further upstream of the CD14 gene is critical for sCD14 expression in hepatocytes but not in monocytes.9 In response to inflammation and infection, IL-6 induces hepatic sCD14 expression; therefore sCD14 is regarded as an acute phase protein.10 Like acute phase proteins, sCD14 levels increase in the presence of acute and chronic infectious and inflammatory diseases, including atherosclerosis. Similar to other acute phase inflammatory biomarkers, sCD14 predicts incident cardiovascular disease and strongly and independently predicts all-cause mortality in older adults.

Assessment of Association Between CD14 Genotype and CVD in Women's Health Initiative
To assess the association between CD14 SNP genotype and CVD, we used data from a larger nested case-control sample of incident CHD and stroke from the Women's Health Initiative.20 The sample included 3042 myocardial infarction (MI) and 2222 stroke EA cases and 296 MI and 251 stroke black cases. There were 2943 EA and 295 black controls without CHD or stroke. Genotyping and quality control of CD14 SNPs were performed using the ITMAT/Broad/CARe (IBC) array at TGen (Phoenix, Arizona). Logistic regression models were adjusted for traditional risk factors (age, BMI, smoking, diabetes mellitus, hypertension, cholesterol) and principal components to control for population stratification.

Methods
CHS Cohort
The CHSAge, sex, and the first 10 eigenvectors derived from principal components analysis were analyzed. For blacks, sCD14 was analyzed using log-transformed values in the genetic association analyses to assess statistical significance. Means and standard errors were reported from the corresponding untransformed model for comparisons with EAs. Haplotype frequency estimation and association analyses were performed using the haplo.stats package for R. Estimation of sCD14 phenotypic variance explained by genomewide single-nucleotide polymorphisms (SNPs) was performed using the Genome-wide Complex Trait Analysis method, as described in the online-only Data Supplement.

We performed Cox proportional hazards models to test for association between sCD14 (or other inflammation biomarkers) and risk of incident CVD events or mortality. sCD14 and other biomarkers were analyzed continuously to estimate the effects of a 1-SD difference in value, as well as categorically using quintiles. The first quintile represented the reference group. For the continuous analysis, IL-6 and CRP were log-transformed. Persons with adjudicated baseline prevalent disease relevant for each outcome were excluded before Figure.