We also tested for a relation between lifetime prevalence of several chronic diseases and FOXO3A genotype (Table 6). A significant protective relation was found for homozygosity for the GallelewithregardtoprevalenceofCHDandaborderlinerelation for cancer. Finally, we assessed the FOXO3A3 MAF distribution by maximum attained age in all participants combined. The MAF increased markedly with age (Table 7).

Analysis of five candidate genes demonstrated that one gene clearly stood out from the others in terms of a potential human longevity gene—FOXO3A. That this gene might be important to human longevity is supported by several lines of evidence. First, in nested case-control analyses, variation within this gene was strongly associated with longevity. Furthermore, two copies of the G allele conferred about twice the protective effect (suggesting an additive

Table 3. FOXO3A3 genotype by case–control status

FOXO 3A3 Genotype (rs 2802292)

Table 4. Difference in health status between genotype groups at baseline

Case-Control Status TT TG GG Average-Lived Phenotype* 223 (55%) 153 (38%) 26 (6%) Longevity Phenotype† 81 (38%) 106 (50%) 26 (12%) p value for Pearson Exact test‡ 0.000091 p value after Bonferroni adjustment 0.00135

Healthy at baseline,* %

Healthy at baseline,* % P value for trend Homo. Homo. Adj. for Major Heter. Minor Unadjusted Case-Control Stat FOXO3A1 57.41 69.48 75.51 0.01 0.065 FOXO3A2 57.37 69.35 77.08 0.01 0.035 FOXO3A3 57.89 68.34 75.00 0.02 0.097

*Number and percentage of subjects from n   402 (cid:6)average-lived(cid:6) decreased controls (mean attained age 78.5 years). †Number and percent of subjects from n  213 (cid:6)long-lived(cid:6) cases (mean attained age 97.9 years). ‡From the exact Pearson (cid:1)2 test comparing the genotype frequencies in the cases and controls.

*(cid:6)Healthy(cid:6) is defined as absence of 6 major chronic diseases (CHD, stroke, cancer, PD, COPD and treated type 2 diabetes; high physical function (can walk one-half mile) and high cognitive function (CASI score   74).

Table 7. Genotype distribution by maximum attained age

Age at death (years)* n MAF of FOXO3A3 72–74 17 0.21 75–79 277 0.25 80–81 108 0.26 95–99 185 0.37 100–106 28 0.39

TT TG GG P* Fasting glucose, mg/dl Average-lived 118.4  34.0 117.4 It 38.0 115.9  40.1 0.80 Long-lived 108.3  20.7 109.1  23.7 110.5  24.1 0.73 Fasting insulin, mIU/liter Average-lived 23.7  81.2 30.4 Ib 91.9 13.2 It 5.9 0.004 Long-lived 13.5   9.0 14.1  13.4 13.3  9.3 0.77 Log fasting insulin, mIU/liter Average-lived 2.68  0.67 2.73  0.85 2.47  0.48 0.03 Long-lived 2.45   0.55 2.43  0.61 2.44   0.52 0.99 HOMA IR Score Average-lived 9.1  53.0 10.0 It 32.2 3.8 It 2.4 0.03 Long-lived 3.7   2.8 4.0  4.3 3.6 14 2.2 0.55

*Thirty-seven cases were still alive; mean age 98.7 years (range 97–106).

also had metabolic profiles suggesting higher insulin sensitivity at younger ages, with lower waist to hip ratio, lower glucose, insulin, and HOMA values (Tables 1 and 5). Several phenotypes were associated with variation in FOXO3A genotype.

Surprisingly, there was no significant difference in diabetes prevalence between cases and controls. However, since the cases were more than a decade older than controls, and diabetes tends to increase markedly with age, it is noteworthy that prevalence of diabetes was not significantly different. In fact, both cases and controls had a high prevalence of diabetes (near 60%), despite relatively low BMI. Why Type 2 diabetes tends to be more prevalent in Japanese at a relatively low BMI is not completely understood (50).However,theremaybemetabolicdifferencesinJapanese(and some other Asians) with higher visceral fat in Asians at lower BMI than in whites and blacks (51, 52). Indeed, Japan national guidelines reflect such population differences and consider Japanese obese at a BMI of 25 (53). Other contributing factors to the high prevalence of diabetes in the HHP/HAAS cohort are that participants were tested for diabetes by several clinical methods and at several examinations making detection more likely.

*P value for Student’s t test comparing mean values between GG genotype and other groups within cases and controls.

effect), roughly tripling the odds of living close to a century. minor allele frequency also rose markedly from septuagenarian centenarian ages (Table 7).

Second, all three SNPs that we assessed in the FOXO3A gene, which were in tight LD, were strongly correlated with the longevity phenotype. This indicates that the finding was unlikely to be due to chance. Third, carriers of the minor (G) alleles were healthier at the   baseline examination, 15 years prior (Table 4).

In fact, the baseline examination suggested that cases were markedly healthier than controls despite the fact that cases were, on average, 11 years older. The cases possessed significantly less age-related disease, including less prevalent CHD, stroke, and cancer. They also had better self-rated health and generally had high physical function, including less difficulty walking. Interest- ingly, despite being more than a decade older than controls, the longevity cases had similar levels of cognitive function. This sup- ports the existence of a ‘‘healthy aging’’ phenotype where individ- uals somehow delay or avoid major clinical disease and disability until late in life. The healthy aging phenotype that we observed in cases is similar to the healthy aging phenotypes reported in cente- narians at younger ages when compared to their age-matched birth cohorts (46–48) and in centenarian offspring (49). Long-lived cases