2.3. Study of Osteoporotic Fractures (SOF)

The SOF cohort served as an additional validation population for this study. The original SOF cohort comprised n = 9704 women of EA descent age 65 and older recruited between September 1986 through October 1988 from 4 U.S. sites: Baltimore, Maryland; Monongahela Valley, Pennsylvania; Minneapolis, Minnesota; and Portland, Oregon (Cummings et al., 1995). The study protocol was approved by the institutional review committee and written informed consent was obtained from all participants. The cohort has been followed for over 20 y with complete mortality follow- up and included 896 SOF participants selected for inclusion in a longevity case-control study (293 long-lived cases P92 y, mean = 95.3 ± 2.1 y and 603 shorter-lived controls age at death 679 y, mean = 75.7 ± 2.6 y).

2. Materials and methods

In order to test our hypothesis in the larger candidate gene pa- nel, we utilized stored DNA and previously measured and collected data from CHS. CHS is a prospective, population-based cohort study of 5888 adult men and women 65 y and older recruited from four field centers: Forsyth County, North Carolina; Sacramento County, California; Washington County, Maryland; and Pittsburgh, Pennsylvania (Fried et al., 1991). Baseline examination for the ori- ginal cohort, of whom 4925 or 95% self-identified their ethnicity as white, was performed over 1 y beginning in May 1989. A second CHS cohort of African–American (AA) participants (n = 687) was re- cruited between 1992 and 1993. All procedures were conducted under institutionally approved protocols for use of human subjects. Medication and lifestyle histories, physical examinations and

2.4. Gene and SNP selection

Our primary hypotheses were that one or more common SNPs in genes from pathways related to inflammation and other stress-responses related to aging were associated with (a) circulat- ing IL-6 levels; (b) longevity case-control status. Candidate genes were chosen based on their known influence on acute and chronic inflammation and involvement in components of IL-6 regulated in- nate immunity, including pathogen recognition, cytokine signaling, NFjB activation, and the generation of acute phase reactants. The complete list of genes is shown in Supplementary Table 1. Within each candidate gene, ‘‘tag” SNPs were selected to capture common linkage disequilibrium patterns using the SeattleSNPs candidate gene SNP discovery resource (http://pga.mbt.washington.edu/) and/or the HapMap data base (http://www.hapmap.org). A com- prehensive assessment of IL6 and CRP gene polymorphisms, IL-6 levels, and mortality risk in CHS has been reported previously (Walston et al., 2007; Hindorff et al., 2008); therefore we did not include IL6 or CRP tagSNPs in the current candidate gene screening analysis. TagSNPs were selected separately in EA and AA using the pair-wise LD binning procedure implemented in the LDSelect algo- rithm of Carlson et al (2004), such that all known common variants [defined as minor allele frequency (MAF) P5%] are correlated with at least one tagSNP with r2 > 0.8. For some LD bins, multiple tag- SNPs were typed in case of assay failure. These redundant tags

Medication and lifestyle histories, physical examinations and phlebotomy samples were obtained at the baseline exam. Phlebot- omy and blood processing methods were described previously (Cushman et al., 1995). Baseline IL-6 was measured from stored plasma samples in 4517 self-identified EA and 829 self-identified AA using a highly sensitive ELISA (Quantikine HS Human IL-6 Immunoassay; RD Systems, Minneapolis, MN). The analytical coef- ficient of variation was 6.3% (Jenny et al., 2002). Of the participants who had baseline IL-6 levels measured, 4190 EA and 766 AA con- sented to genetic testing and had DNA aliquots available for genotyping.

Complete follow-up data for the CHS cohort were available through June 2005. Fatal events due to all-causes were defined and adjudicated by physician review, according to medical records and death certificates. The National Death Index provided com- plete mortality follow-up. Cause of death was adjudicated by using published criteria: cardiovascular disease (CVD) death was defined as death due to atherosclerotic coronary heart disease, cerebrovas- cular disease, atherosclerotic disease, or other CVD (Ives et al., 1995). For our candidate gene – longevity phenotype association analysis, we defined longed-lived cases as subjects who lived to at least 92 y of age (n = 479; 61% female) and shorter-lived ‘‘con- trols” as subjects who died before the age 79 y (n = 900; 46% female).

didate genes by the Center for Inherited Disease Research (CIDR) using the Illumina GoldenGate 1536 SNP platform. Overall geno- type missing rate was <0.1%; genotyping failure rate did not exceed 4.2% for any of the SNPs under study. Concordance rates for blind duplicate samples was >99.5% for all SNPs assays across both geno- typing platforms. We excluded SNPs from analysis with minor al- lele frequency less than 5%. Using this procedure, we identified 477 and 727 tagSNPs bins among EAs and AAs, respectively, across 87 candidate genes. Of the total number of tagSNPs analyzed (n = 791), 413 or 52% were common to both EA and AA, while 64 and 314 were unique to EA and AA, respectively.

2.2. Centenarian case-control study of Ashkenazi Jews

In order to validate significant results identified in the CHS pop- ulation, we utilized previously collected DNA and phenotypic data from a study of three hundred and eighty eight Ashkenazi Jewish cases with exceptional longevity [289 females and 99 males, aver- age age 97.7 (0.2) years (mean SE), age range 95–108 y; 20% over the age of 100] and Ashkenazi Jewish control subjects [n = 364, mean age 79.5 (0.4) years (mean SE), 58% female] without a family