Human longevity, however, is a complex phenotype that encom- passes disease-specific risks as well as the individual rate of aging. The study of its genetic antecedents is challenging. The study of longevity may be affected by small genetic effect sizes, population stratification artifact, population heterogeneity, lack of sufficient numbers of long-lived study participants, and other problems (3, 4, 41). Therefore, to assess potential genetic contributions to human longevity from genes linked to IIS signaling, we chose a large, homogeneous, long-lived population of men well-characterized for aging phenotypes, and we performed a nested-case control study of 5 candidate longevity genes with links to the IIS pathway. These genes were chosen based on prior associations with aging pheno- types principally from gene knockout, transgenic, mutant, and other model organisms (3, 4, 14–17, 36, 42). Priority was for genes involving insulin sensing and glucose (energy) homeostasis.

Five genes were investigated (ADIPOQ, FOXO1A, FOXO3A, SIRT1, and COQ7). Minor allele frequencies and other related genetic information for the cases and controls are presented in Table 2. However, only FOXO3A genotype was associated with om<I longevity using an initial cut-off value of P 0.05.

Further investigation comparing the genotype frequencies of FOXO3A3 between cases and controls revealed a highly significant difference P   0.00009 for the Pearson’s exact (cid:1)2 statistic (Table 3). Five loci with 3 SNPs within each allele were tested (Table 2). Bonferroni adjustment for multiple comparisons resulted in a corrected p value of 15 xX | 0.00009   0.00135. Due to the high LD between the 3 SNPs of FOXO3A, we further investigated the FOXO3A3 SNP only (rs 2802292). The OR for homozygous minor vs. homozygous major alleles for FOXO3A3 between the cases and controls was 2.75 (95% CI: 1.51  5.02, P ar)(  0.0007), and the OR for heterozygous vs. homozygous major alleles between the cases and controls was 1.91 (95% CI: 1.34 | 2.72, P Ue 0.0003). These results suggest an additive effect on longevity.

Results

The baseline characteristics of the HHP/HAAS study population at the 1991–1993 examination are presented in supporting informa- tion (SI) Table S1. The mean age was 77.9 years and 100% of the population was male and of Japanese ethnicity. Biological charac-

Table 2. Candidate genes for human longevity and the MAF in cases and controls

Gene name Symbol SNP ID name Cases Controls P* Adipo,† C1Q, CDC ADIPOQ rs1063539 ADIPOQ(cid:1)1 0.297 0.263 0.20 rs182052 ADIPOQ(cid:1)2 0.455 0.493 0.22 rs266729 ADIPOQ(cid:1)3 0.195 0.239 0.08 Forkhead Box O1A FOXO1A rs2755209 FOXO1A1 0.272 0.291 0.48 rs2721069 FOXO1A2 0.293 0.307 0.62 rs2755213 FOXO1A3 0.350 0.358 0.77 Forkhead Box O3A FOXO3A rs2764264 FOXO3A1 0.347 0.248 0.0002 rs13217795 FOXO3A2 0.340 0.248 0.0006 rs2802292 FOXO3A3 0.371 0.255 (cid:2)0.0001 Sirtuin 1 SIRT1 rs7069102 SIRT1(cid:1)1 0.185 0.181 0.84 rs10823112 SIRT1(cid:1)2 0.337 0.360 0.44 rs1885472 SIRT1(cid:1)3 0.188 0.179 0.71 Coenzyme Q7 COQ7 rs8051232 COQ7(cid:1)1 0.147 0.150 0.90 rs11074359 COQ7(cid:1)2 0.153 0.171 0.43 rs7192898 COQ7(cid:1)3 0.162 0.170 0.73

MAF between cases and controls with (cid:1)2 test. C1Q, and collagen domain containing.

Discussion

To understand more about the longevity phenotype at younger ages, we compared the proportion of people who were healthy at the baseline examination (1991–1993) for each of the three FOXO3A genotype groups using the definition of healthy survival from Willcox et al. (44). The differences were highly significant (Table 4). Those who possessed one or more G alleles were more likely to be healthy at baseline than those who were homozygous for the major (TT) allele; 75% of those homozygous for the minor allele were healthy at the baseline examination vs. 57% of those homozygous for the major allele. After adjusting for case-control status, the differences were still marginally significant. This suggests remaining association of the allele with health status in cases and controls.

The rapid aging of the population will place unprecedented disability (45). Better understanding of mechanisms of including biological pathways that may have widespread on how we age, could have important implications for lowering risk for age-related disease and disability. There are many ically plausible candidate genes for human longevity but only finding has so far been widely replicated in multiple that of the APOE gene (3). This gene has widespread effects and as such influences the ability to achieve a long and healthy

To find other such genes, it may be helpful to use model organisms to identify a priori potential candidates before conduct- ing human studies. Therefore, we chose to study several candidate genes within the human insulin/IGF-1 signaling pathway and/or oxidative stress response system on the basis of sequence and/or functional homology with model organisms of aging or prior human studies. We constructed a list of human candidate genes (Table S2) fromthesesignalingpathwaysandassessedvariationsinthesegenes   occurring at a frequency of 10% in the Japanese population. Three SNPs were chosen from each gene for analysis. SNPs were selected mainly from regions with linkage disequilibrium (LD) for maximal coverage of each gene.

Toassesswhethertherewasarelationbetweeninsulinsensitivity, a potential intermediate phenotype of longevity, and genotype, we tested the relation between fasting insulin, glucose, HOMA and genotype (Table 5). For non-normally distributed variables we used log conversion to a normal distribution. There was a significant relation between insulin, log insulin, HOMA and genotype. Ho- mozygosity for the G allele was associated with markedly lower insulin, log insulin and HOMA score, but in controls only.