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

Alex P. Reiner, Ethan M. Lange, Nancy S. Jenny, Paulo H.M. Chaves, Jaclyn Ellis, Jin Li, Jeremy Walston, Leslie A. Lange, Mary Cushman, Russell P. Tracy

Objective—CD14 is a glycosylphosphotidylinositol-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 glycosylphosphotidylinositol 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 ◼ glycosylphosphotidylinositol ◼ mortality

CD14, sCD14 is 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 concentra- tions, LPS binding protein and sCD14 downregulate LPS- induced responses13 by transfer of LPS to lipoproteins for subsequent removal.

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 solu- ble form (sCD14).4 In addition to secretion from liver, sCD14 may be derived by enzymatic cleavage of GPI-anchored cell membrane-bound CD14.4–9

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 ing sCD14 is regarded as an acute phase protein.10 Like acute phase proteins, sCD14 levels increase in the of acute and chronic infectious and inflammatory including atherosclerosis. Similar to

From the Department of Epidemiology, University of Washington, Seattle WA (A.P.R.); Department of Genetics, University of North Carolina, Chapel Hill, NC (E.M.L., J.E., J.L., L.A.L.); Department of Medicine, Family Center for Geriatric Research and Education, Herbert Wertheim College of Medicine, Florida International University, Miami, FL (P.H.M.C.); Department of Medicine and Pathology, University of Vermont, Burlington, VT (N.S.J., M.C., R.P.T.); and Department of Medicine, Johns Hopkins School of Medicine, Baltimore, MD (J.W.).

The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.112.300421/-/DC1. Correspondence to Alex Reiner, Department of Epidemiology, Box 357236, University of Washington, Seattle, Washington 98195. E-mail apreiner@u. washington.edu © 2012 American Heart Association, Inc.

Despite its potential importance for atherosclerosis and other aging-related inflammatory diseases, sCD14 has not been well characterized in prospective, population-based studies of older adults. We therefore measured and analyzed sCD14 in the Cardiovascular Health Study (CHS), a large cohort of older adults with follow-up for incident cardiovascular disease (CVD) and mortality for up to 20 years. We first characterized the cross- sectional relationship of sCD14 to other traditional CVD risk factors and subclinical vascular disease, as well as other inflam- matory CVD biomarkers (fibrinogen, C-reactive protein [CRP], and IL-6). Second, we conducted genomewide and CVD can- didate gene-centric association scans of sCD14 in the CHS, separately in European-Americans (EA) and blacks. Finally, we assessed the ability of sCD14 and its main genetic determinants to predict incident CVD and mortality during follow-up.