4. Estimated Hazard Ratios for Association Between 1 SD Unit Increase in Soluble CD14 and Incident CVD Events and

Mortality (n=3820) MI (n=870) CHD (n=1547) Stroke (n=945) Model HR (95% CI) P Value HR (95% CI) P Value HR (95% CI) P Value HR (95% CI) P Minimal (1) 1.25 (1.21–1.29) 1.2×10–42 1.10 (1.03–1.19) 0.006 1.14 (1.09–1.20) 2.4×10–7 1.15 (1.07–1.23) Multivariable (2) 1.11 (1.07–1.15) 5.5×10–9 1.07 (0.99–1.15) 0.075 1.11 (1.06–1.18) 7.5×10–5 1.12 (1.04–1.20) Subclinical (3) 1.09 (1.06–1.13) 1.3×10–6 1.03 (0.96–1.11) 0.38 1.07 (1.01–1.13) 0.017 1.11 (1.03–1.19) Biomarkers (4) 1.09 (1.05–1.13) 2.9×10–5 0.96 (0.89–1.05) 0.39 1.01 (0.95–1.07) 0.73 1.08 (1.00–1.17)

CVD indicates cardiovascular disease; MI, myocardial infarction; CHD, coronary heart disease; lipoprotein; and HR, hazard ratios. Model definitions:

) Age, sex, race.

) Model

+smoking, diabetes mellitus, hypertension, SBP, LDL (baseline CVD, cystatin C).

) Model

+carotid IMT and ankle-brachial index.

) Model

+C-reactive protein, interleukin-

, and fibrinogen.

and rs778584 were each nominally associated with 1.3- to 1.6-fold increased risk of stroke in black, whereas rs778584 was associated with decreased risk of CHD in EA (P=0.025). However, these results were not significant when corrected for multiple hypothesis testing. Nor was there significant evidence for association between CD14 genotype and risk of CHD or stroke in a second, large case-control sample from the Women’s Health Initiative (Table IV in the online-only Data Supplement).

within several putatively functional genomic elements indica- tive of chromatin accessibility, including an Encyclopedia Of DNA Elements Consortium (ENCODE) DNaseI hypersensi- tivity site, histone methylation and acylation enrichment sites, and hepatocyte-specific transcription factor binding elements for hepatocyte nuclear factor 4 alpha (HNF4A)- and forkhead box protein A1 (FOXA1), as determined by genomewide chromatin immunoprecipitation (ChIP)-Seq.22

Two other CD14 variants (haplotypes 2 and 3 in Table 3) were associated with higher sCD14 levels. Haplotype 2 (tagged by rs778584 in our data set) contains the previously characterized –159 T allele of rs2569190, which is located in the proximal promoter.23 The CD14 –159 T allele poly- morphism increases transcription of CD14 in monocytes by decreasing binding affinity to the inhibitory Sp3 subunit of an Sp transcription factor site15 and has been associated with higher sCD14 concentration14 as well as greater density of mCD14 in some studies24 but not in others.25,26

Discussion

In a biracial population of older adults, we have character- ized the epidemiological and genetic associations of sCD14, as well as relationships of this inflammatory biomarker to incident CVD and mortality. Baseline sCD14 was correlated positively with EA ethnicity, female sex, traditional CVD risk factors (smoking, hypertension, diabetes mellitus), and other inflammatory biomarkers (IL-6, CRP, fibrinogen), and negatively correlated with BMI. We identified 2 genomewide significant sCD14-associated loci, the CD14 structural locus on chromosome 5q21 and a novel missense variant of PIGC, which encodes an enzyme required for the first step in GPI anchor biosynthesis. We also showed that sCD14 predicts incident CVD and mortality in older adults.

levels of other commonly measured inflammation biomarkers among blacks, we found that levels of sCD14 were lower in blacks than EA. Whether the ethnic differences in sCD14 are a result of environmental and genetic factors is uncertain. The CD14 gene locus shows evidence of balancing selection, par- ticularly among European populations,27 which could be attrib- utable to its impact on the magnitude of host immune response to local pathogens. It is also noteworthy that the 2 CD14 alleles tagged by rs778584 and rs4914 associated with higher sCD14 are both more common in EA than blacks, whereas the sCD14- lowering haplotype tagged by rs5744451 is common only among blacks. Assuming independence of SNPs, and an addi- tive genetic model, differences in allele frequencies between the identified SNPs accounts for ≈23% of the interpopulation differences in sCD14 levels. Therefore, allele frequency differ- ences at the CD14 locus provide some potential explanation for the higher sCD14 levels observed in EA.

identified intra- and interpopulation allelic heterogeneity at the CD14 locus, including 2 new CD14 variants associated with lower sCD14 levels. The significant, nonredundant vari- ants at the CD14 locus explain 3.7% of the trait variability in EA versus 9.0% in blacks. One of these variants (haplo- type 4 tagged by rs5744455 in Table 3) was associated with lower sCD14 in both EA and blacks. rs5744455 may represent a functional variant, as this SNP is located ≈50 bp upstream of the transcription start site within an ENCODE DNaseI hypersensitivity site in CD14+ monocytes and promyelocytic cells. DNaseI hypersensitivity sites are characterized by open chromatin and tend to be transcriptionally active. The other newly discovered sCD14-lowering variant was present only among blacks (haplotype 5 tagged by rs5744451 in Table 3. Of the SNPs associated with haplotype 5, rs5744430 lies within a 700-bp hepatocyte-specific enhancer region located ≈6 kb 5ʹ of the transcription start site.9,21 rs5744430 is also located

CD14 is 1 of >100 cell-surface proteins anchored to the plasma membrane via GPI. Assembly of GPI-linked proteins occurs in the endoplasmic reticulum and involves more than 30 different genes.28 The first step of the biosynthesis, the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to phosphatidylinositol, is mediated by at least 3 genes (PIGA,