For the Ashkenazi Jewish validation study, genotyping was per- formed at the Albert Einstein College of Medicine using Sequenom MASSarray(cid:3) System (Sequenom Inc., San Diego, CA) according to the manufacturer’s instructions. SOF validation genotyping was performed using the SNPstream 48plex genotyping platform

(Beckman Coulter, Fullerton CA) according to manufacturer’s instructions.

3. Results

2.5. Statistical analysis

CHS participant baseline characteristics by race are summarized in Table 1. In univariate and multivariable-adjusted regression analyses, older age, male sex, current smoking, higher BMI, alcohol consumption, and prevalent CVD were significantly associated with higher IL-6 (all p < 0.001). When analyzed by quartiles, and adjusted for other risk factors, baseline IL-6 levels were strongly and independently associated with a linear increase in all-cause mortality, as well as mortality due to cardiovascular and non-car- diovascular causes (Table 2).

To assess the association of baseline plasma IL-6 concentration with mortality during follow-up, we used Cox proportional hazards to model quartiles of IL-6 level. Results were reported as hazard ra- tios with 95% confidence intervals (95%CI). Hazard ratios were ad- justed for baseline age, sex, race, body mass index, total cholesterol, diabetes, systolic blood pressure, smoking, race, clinic site, and clinical CVD at baseline.

each SNP and logarithmically transformed IL-6 concentration lev- els using multiple linear regression that included covariate terms for age, sex, clinic, body mass index, and smoking. Analyses were performed separately in CHS EA and AA participants. SNP genotype was coded 0/1/2 for the number of copies of the minor allele assuming an additive genetic model. Logistic regression was used to assess associations between each SNP genotype and longevity case/control status. Cox regression was used to model associations between SNP genotype and total mortality or cause-specific mor- tality (cardiovascular or non-cardiovascular). Covariates for the initial case-control longevity analysis included sex, and recruit- ment site. The Cox models were also adjusted for baseline age. We also performed more fully adjusted multivariable models that included baseline hypertension, diabetes, smoking status, clinical CVD, and diagnosis of cancer. Clinical CVD, used for adjusting multivariable models, was defined as self-reported myocardial infarction, stroke, angina, transient ischemic attack, claudication, or re-vascularization procedure.

3.2. Candidate gene SNP associations with IL-6 phenotype in CHS

Using a false discovery rate threshold of 5%, three tagSNPs with- in the IL6R gene on chromosome 1q21.3 [rs4537545 (q- value < 10(cid:2)15), rs4553185 (q-value < 10(cid:2)8), and rs4072391 (q- value < 10(cid:2)3)] and one tag SNP within the PARP1 gene on 1q42.12 (rs1805415; q-value < 0.01) were significantly associated with baseline IL-6 concentration in EA (Table 3). IL6R encodes a subunit of the IL-6 receptor complex, while PARP1 encodes the DNA repair enzyme poly-ADP ribose polymerase. While both genes are located on the short arm of chromosome 1, there was no evi- dence of linkage disequilibrium between IL6R and PARP1.

The three IL6R tagSNPs most strongly associated with IL-6 phe- notype were moderately correlated with one another in CHS EA (pair-wise r2 between rs4537545 and rs4553185 = 0.44; rs4537545 and rs4072391 = 0.15; rs4553185 and rs4072391 = 0.26). When IL-6 phenotype was regressed simultaneously on these three IL6R tagSNPs, only rs4537545 remained statistically significant; each additional copy of the rs4537545 minor allele was associated with a 20% increase in mean log IL-6 levels

In the CHS AA, genotype data were also available for a genome- wide set of 24 unlinked ancestry-informative SNP markers, which were used to adjust for European admixture and population strat- ification in AAs (Reiner et al., 2005). To examine the potential ef- fects of within-Europe population stratification in CHS, we repeated our analysis for any candidate gene SNP associated with IL-6 or longevity following correction for population stratification. Specifically, we computed a Bayesian model-averaged population structure estimate from 1038 autosomal SNPs typed in CHS (excluding SNPs from pairs with r2 > 0.8), and then assigned indi- viduals to six genetically similar clusters (Corander and Tang, 2007). Genetic background cluster was then used as an adjustment variable in regression models assessing the association between candidate gene SNP and phenotype. A subsequent generalized analysis of molecular variance showed that these clusters ex- plained a significant proportion (6.6%, p < 0.001) of the genetic background among CHS European–Americans (Nievergelt et al., 2007).

Table 1

Baseline characteristics of European–American and African–American CHS participants.

Characteristic European–Americans Number 4190 766 Mean age, years (range) 73 (65–98) 73 (65–92) Female sex 1064 (57) 202 (62) Current smokers 206 (11) 50 (15) Body mass index (kg/m2) 26.3 ± 4.5 28.4 ± 5.3 Total cholesterol (mg/dL) 212 ± 40 210 ± 37 HDL cholesterol (mg/dL) 54 ± 16 59 ± 17 Triglycerides (mg/dL) 141 ± 75 116 ± 69 Systolic blood pressure (mm Hg) 136 ± 22 142 ± 23 Diagnosis of hypertension 1020 (55) 239 (73) Diabetes 277 (15) 74 (23) Prevalent CVDa 315 (17) 52 (16) IL-6 (pg/mL) 240 ± 541 374 ± 830