To assess the overall statistical significance and correct for mul- tiple SNP comparisons, we used the false discovery rate (FDR) to control the type I error (Storey and Tibshirani, 2003). The FDR pro- cedure provides an estimate of significance in terms of the q-value, the proportion of significant features expected to be false among the total # of tested SNPs. Results were considered statistically sig- nificant if q < 0.05. SNPs found to be nominally associated with lon- gevity in CHS (p < 0.02) were genotyped in SOF and the Ashkenazi Jewish centenarian case-control studies.

Data are presented as number (%) or mean ± standard deviation, unless indicated.

a Self-reported myocardial infarction, stroke, angina, transient ischemic attack, claudication, or re-vascularization procedure.

Table 2

IL-6 concentration and subsequent risk of mortality in CHS.

Summary effect estimates for longevity status were calculated under an additive genetic model using a meta-analysis approach with inverse-variance weighting. Meta-analysis was performed across all three studies using either a fixed-effects or random-ef- fects model (DerSimonian and Laird, 1986) with the STATA soft- ware package v.8.2 (Stata Corporation, College Station, Texas). The random-effects model allows for heterogeneity of results be- tween studies.

IL-6 level All-cause Hazard CVD Hazard ratio Non-CVD Hazard ratio (95% CI) (95% CI) ratio (95% CI) <1.2 pg/mL 1 1 1 1.2–1.7 pg/mL 1.34 (1.17–1.52) 1.61 (1.29–2.01) 1.19 (1.01–1.41) 1.8–2.6 pg/mL 1.71 (1.49–1.94) 2.07 (1.67–2.58) 1.52 (1.29–1.75) P2.6 pg/mL 2.34 (2.05–2.65) 2.65 (2.14–3.30) 2.17 (1.85–2.56) P for trend 5   10(cid:2)43 1    10(cid:2)20 6  10(cid:2)24

Adjusted for age, race, sex, CVD, cancer, smoking, hypertension, diabetes, and

Table 3

SNPs associated with plasma IL-6 concentration in CHS European–Americans.

SNP rs# Gene Chr Position (bp) Location Minor allele Correlated SNP r2 with reference SNP Regression coefficient p-Value frequency ± standard errora rs4537545 IL6R 1 152685503 Intron 0.41 Tag SNP – 0.114 ± 0.013 3 3x 1 10(cid:2)18 rs7518199 IL6R 1 152674043 Intron 0.41 Typed SNP 0.88 0.101 ± 0.013 3 3x 1 10(cid:2)18 rs8192284 IL6R 1 152693594 Ala358Asp 0.35 HapMap SNP 0.96 – – rs4553185 IL6R 1 152677579 Intron 0.43 Tag SNP – (cid:2)0.082 ± 0.013 3 3x1 10(cid:2)10 rs4845618 IL6R 1 152666639 Intron 0.43 Typed SNP 0.96 (cid:2)0.078 ± 0.013 3 3x1  10(cid:2)10 rs4072391 IL6R 1 152705504 30 UTR 0.19 Tag SNP (cid:2)0.076 ± 0.017 5   10(cid:2)6 rs2229238 IL6R 1 152704520 30 UTR 0.19 Typed SNP 0.98 (cid:2)0.079 ± 0.017 5  10(cid:2)6 rs1805415 PARP1 1 224637463 Lys352Lys 0.15 Tag SNP – 0.095 ± 0.025 1 1x 1 10(cid:2)4 rs752307 PARP1 1 224618152 Intron 0.16 Typed SNP 0.997 0.067 ± 0.018 2 2x 1 10(cid:2)4 rs1136410 PARP1 1 224621925 Val762Ala 0.17 HapMap SNP 1.00 – –

Adjusted for age, sex, clinic, BMI, and smoking. a Regression coefficient equals the estimated change in log(IL-6) level per each additional copy of the minor allele.

a Regression coefficient equals the estimated change in log(IL-6) level per each additional copy of the minor allele.

(p-value < 10(cid:2)15). Notably, IL6R rs4537545 tags another IL6R SNP rs8192284, which encodes a functional Ala358Asp polymorphism (Table 3). The minor allele of PARP1 rs1805415 (which encodes a synonymous coding SNP) was associated with (cid:4)18% increase in mean log IL-6 levels. PARP1 rs1805415 is in strong linkage disequi- librium with rs1136410, which encodes a non-synonymous Va- l762Ala amino acid substitution. Overall, IL6R genotype explained 2% of the inter-individual variation in IL-6 phenotype among EA, while PARP1 genotype explained 0.2%. By comparison, age, sex, clinic, BMI, and smoking explained 12% of the IL-6 phenotypic variance.

(odds ratio = 0.75; 95%CI 0.56–0.99; p = 0.04). In contrast, there was no association between PARP1 rs1805415 among SOF longev- ity cases and controls (odds ratio = 1.02; 95%CI 0.77–1.36; p = 0.88). There was no evidence that the association between PARP1 rs1805415 and longevity differed by gender in either CHS or the Ashkenazi Jews (data not shown). In the pooled analysis of all three studies, the risk of longevity associated with the minor al- lele of PARP1 rs1805415 was 0.79 (95%CI 0.67–0.92; p = 0.003) un- der a fixed-effect model. Under a random-effect model, the pooled odds ratio was 0.79 (95%CI 0.62–1.02; p = 0.07), with an estimate of between-study variance of 0.03. Seven other SNPs nominally asso- ciated with longevity in CHS (p < 0.02) showed little evidence of association with longevity in either SOF or the Ashkenazi Jewish centenarian study (Supplemental Table 4).

In EA, tagSNPs within several genes encoding components of the nuclear factor kappa B (NFjB) transcription factor complex (IKBKE, IKBKB, and RELA) or the IL-6 signal transducer and activator of transcription-3 gene (STAT3) showed weaker evidence of associ- ation with IL-6 phenotype (Supplemental Table 2). Among the smaller CHS African–American cohort, an intronic SNP within the Toll-like receptor 4 gene (TLR4) had a borderline association with higher IL-6 levels (q-value = 0.07). Several additional tagSNPs of IL6R, STAT3, or IL6ST (which encodes the IL-6 signal transducer component of the IL-6 receptor complex) showed weaker evidence of association with IL-6 phenotype among AAs (Supplemental Ta- ble 3).

4. Discusssion