The human homologue of DAF-16 includes four FOXOs: FOXO1, FOXO3, FOXO4 and FOXO6. We hypothesize that common, natural variation in the form of single nucleotide poly- morphisms (SNPs) in FOXO and related genes might influence human aging and longevity. A connection between insulin, FOXO, oxidative stress, and human longevity would be particularly inter- esting since oxidative stress has long been a favorite putative mechanism of aging. Since 1956, the free radical theory of aging has hypothesized that aging results partly from damage to DNA, cells, and tissues from cumulative exposure to reactive oxygen molecules

Author contributions: B.J.W., T.A.D., Q.H., R.C., J.S.G., K.Y., K.H.M., D.C.W., B.R., and designed research; B.J.W., T.A.D., Q.H., R.C., J.S.G., K.Y., K.H.M., D.C.W., B.R., and J.D.C.

Author contributions: B.J.W., T.A.D., Q.H., R.C., J.S.G., K.Y., K.H.M., D.C.W., B.R., and J.D.C. designed research; B.J.W., T.A.D., Q.H., R.C., J.S.G., K.Y., K.H.M., D.C.W., B.R., and J.D.C. per- formedresearch;T.A.D.contributednewreagents/analytictools;Q.H.,R.C.,andJ.S.G.analyzed data;andB.J.W.,T.A.D.,Q.H.,R.C.,J.S.G.,K.Y.,K.H.M.,D.C.W.,B.R.,andJ.D.C.wrotethepaper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. §Towhomcorrespondenceshouldbeaddressedat:PHRI,846SouthHotelStreet,Suite201, Honolulu, HI 96813. E-mail: bjwillcox@phrihawaii.org. This article contains supporting information online at www.pnas.org/cgi/content/full/

In contrast, there have been several robust genetic findings in model organisms of aging (11–13). For example, variation in single genes can result in substantial differences in lifespan in model organisms, particularly with genes that are considered part of the insulin/IGF-1 (IIS) signaling pathway (14–18).

§Towhomcorrespondenceshouldbeaddressedat:PHRI,846SouthHotelStreet,Suite201, Honolulu, HI 96813. E-mail: bjwillcox@phrihawaii.org.

Mutations that increase SIR-2 activity or that decrease IGF-1 signaling both increase the lifespan of C. elegans by the DAF-16/FOXO protein (19, 20). In mammalian cells, a

© 2008 by The National Academy of Sciences of the USA

(mean attained age 78.5 (mean attained age 97.9 years) (n = 1 402) years)* (n   213) Variables at baseline examination (1991–1993) Mean It  SD Min–Max Mean It  SD Min–Max P† Biological, fasting values Age at baseline exam, years 74.63 It 2.05 71–79 85.62  3.12 80–93 (cid:2).0001 Body mass index, kg/m2 23.4 I 3.17 15.89–32.33 23.0  2.91 15.4–31.1 0.1272 Waist/hip ratio 0.95   0.06 0.78–1.15 0.93  0.06 0.73–1.07 0.0008 Total cholesterol, mg/dl 187.96 +1 34.6 98–303 185.36   32.16 95–304 0.3680 HDL, mg/dl 50.82  14.17 21–129 51.29  13.54 27–100 0.6911 Triglycerides, mg/dl 154.72  118.72 46–1369 140.32  82.23 38–649 0.1178 Log triglycerides‡ 4.88  0.51 3.83–7.22 4.81  0.50 3.64–6.48 0.0965 Glucose, mg/dl 117.83 tI 35.9 69–323 108.98   22.55 77–298 0.0012 Insulin, mIU/liter 25.54  82.89 3.3–1164 13.8  11.39 1.5–104 0.0421 Log Insulin‡ 2.69  0.74 1.19–7.06 2.44  0.58 0.41–4.64 (cid:2)0.0001 FOXO3A3 MAF (rs2802292)§ 0.255 — 0.371 — (cid:2)0.0001 General Health Status Self-rated (cid:6)poor(cid:6) health, % 41.92 — 31.07 — 0.0163 Disease prevalence CHD, % 26.37 — 7.55 — (cid:2)0.0001 Stroke, % 7.46 — 3.3 — 0.0394 Cancer, % 20.15 — 13.68 — 0.0468 Diabetes, % 60.55 — 59.81 — 0.8587 Physical/cognitive function Lower body (difficulty walking), % 30.59 — 16.83 — 0.0002 Upper body (grip strength), kg 29.85  7.54 0–47 26.37  5.53 8–45 (cid:2)0.0001 Cognitive score (CASI)§ 80.96   19.48 0–100 78.54  13.85 12–98 0.1088

Variables at baseline examination

*Cases (longevity phenotype) consisted of all HHP/HAAS participants with DNA samples (living and dead) who had reached the age of 95yearsbyAug.2007:Gp1:Alive,n(cid:1)37,meanage98.7,range97–106years;Gp2:dead,n(cid:1)166,meandeathage97.5,range95–106y). †P value from Student’s t test for continuous variables and (cid:1)2 for categorical variables. ‡Log transformation performed for variables not normally distributed. §MAF, minor allele frequency; CASI, cognitive abilities screening instrument.

teristics, general health status, disease prevalence, and functional status are presented.

(33) and although not yet universally accepted, supportive evidence has accumulated over the years (34, 35). Thus, FOXO may provide a potential forkhead or bridge between insulin signaling, free radicals, and human aging/longevity.

From this 1991–1993 baseline population, we selected all partic- ipants who, by 2007, had survived to age 95 years or more as Phy ‘‘longevity’’ cases (n 213). We then selected all participants who pean died before the age of 81 years as ‘‘average-lived’’ controls (n 402). Baseline characteristics of the cases and controls are pre- sented in Table 1. In terms of biological characteristics, the long- lived cases were older, leaner (lower waist:hip ratio), had lower triglycerides (borderline), lower glucose, lower insulin levels, and higher prevalence of the FOXO3A3 allele at the baseline exami- nation. The cases also had better self-rated health and lower prevalence of cardiovascular disease [coronary heart disease (CHD) and stroke] and cancer. Functionally, they appeared better able to walk but had lower grip strength. There was no difference

There has been some prior work linking genes in the IIS pathway to human longevity (36, 37) including an interesting recent report by Suh et al. (38), which links functionally significant IGF-1 receptor mutations to exceptional longevity, but we have not found any published reports of association between FOXO genes and human longevity. Prior studies have found links between FOXO genes and other aging phenotypes, including 4-year survival and stroke risk (39) as well as premature menopause (40).