There are several other strengths to this study. First, the candi- date genes selected for analysis were chosen a priori based on hypothesis-driven criteria. That is, studies of models organisms of aging employing various methods, particularly knockouts, have shown that the IIS pathway is important for aging and longevity. Andmanyfunctionsappeartobeevolutionarilyconserved.Second, the findings are strong, highly significant, and include several

Genotyping. We chose 3 SNPs from each of 5 candidate genes. We chose genes that have well-described influences on aging pathways in model organisms. All geneswerechosenbasedonhypotheticallinkstotheIISpathwayandpotential

linkstoenergyhomeostasis,glucose,and/orlipidmetabolism[seeTableS2].SNPs werechosenbasedontheirminorallelefrequenciesreportedintheHapMapor JSNP database (snp.ims.u-tokyo.ac.jp).

Statistical Analysis. SNPs were evaluated for deviation from Hardy-Weinberg equilibrium. The Pearson (cid:1)2 test was used to compare the cases and controls for equal genotype frequencies using the software program StatXact (68). For esti- mates of strength of association, odds ratios were estimated using logistic re- gression models from SAS (69). General linear model and analysis of covariance were further used to compare proportion of healthy study participants by FOXO3A genotype. For the analysis of aging phenotypes in case and controls, Student’s t test for comparing distribution of continuous variables and (cid:1)2 for proportional variables.

Total cellular DNA was isolated using the PureGene system (Gentra Systems, Inc.) quantified using PicoGreen staining (Molecular Probes) and SNPs from candidategenesgenotypedusingallelicdiscriminationassays.TaqMan(Applied Biosystems) reagents were purchased from ABI and SNPs were chosen with a frequency    0.1 in the Japanese population (www.ncbi.nlm.nih.gov/projects/ SNP).PCRwasamplifiedunderstandardconditionsusingTaqGold(Perkin-Elmer) and detection of PCR products with Taq Man assay, using a 6-FAM-labeled FRET probeforonealleleandaVIC-labeledprobefortheotheralleleandusingminor groove binding (MGB) quenchers to enhance detection of assays. PCR products were measured with the ABI Prism 7000 Sequence Detection System.

ACKNOWLEDGMENTS. We thank Evelyn Hein, Eva Ardo (PHRI), and Sayaka Mitsuhashi (Okinawa Research Center for Longevity Science, Japan) for their helpful research assistance. This study was supported by contract N01-HC- 05102 from the National Heart, Lung, and Blood Institute, contract N01-AG- 4-2149 and Grants 5 U01 AG019349-05, R01 AG027060-01 (Hawaii Lifespan Study), and K08 AG22788-02 from the National Institute on Aging, and Grant 2004-0463 from the Hawaii Community Foundation.

Genotype data were managed through an integrated database system (MS Excel, Microsoft). All positive controls on each genotyping plate were also eval- uated for consistency. Positive markers were tested for deviation from Hardy- Weinberg equilibrium. Call rates exceeded 98%.

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