Table 2 shows the allelic association of FOXO1A and FOXO3A with longevity (sex-combined in population 1). The table includes SNP, allele (major/minor), function, MAF (case/control), P, OR (95% CI), P bP, PW (a<0.05), and HWT. MAF refers to minor allele frequency; P refers to P-value from Hardy-Weinberg equilibrium test; OR (95% CI) refers to odds ratio with 95% confidence interval; P bP is corrected P by Bonferroni step-down method; PW is power to reach an alpha level of 0.05 with a given population size; NC refers to not calculated; 3'-UTR refers to 3-untranslated region.

Table 3 presents gender effects on allelic association of FOXO1A and FOXO3A with longevity trait in population. The table lists SNP, male, female, MAF (case/control), OR (95% CI), P bP, and HWT. MAF refers to minor allele frequency; OR (95% CI) refers to odds ratio with 95% confidence interval; P refers to P-value from x2 test; bP is corrected P by Bonferroni step-down method.

Linkage disequilibrium and haplotype association analysis was performed and presented in J IV 0.90. Haplotypes with a frequency of more than 5% were subjected to an association test with longevity. We tested the distributions of seven selected haplotypes, five from FOXO1A and two from FOXO3A, in female centenarians and controls. The haplotypes TTG and CCG of FOXO1A as well as GTC and CGT of FOXO3A presented significant differences in their distributions between case and control with statistical power greater than 0.96. For FOXO1A, the frequency of haplotype TTG was significantly lower in centenarians than in controls, while CCG showed the opposite. A permutation test was used to correct multiple comparisons. The permutation P-values were 0.024 and 0.046, respectively (Supplementary Material, Table S2). For FOXO3A, GTC lost its statistical difference in permutation analysis. CGT appeared more frequently in centenarians than in controls (permutation P-values less than 0.009; Supplementary Material, Table S2). 

Genotype association analysis was performed to test the inheritance models through which FOXO1A and FOXO3A are associated with longevity. The test was conducted using logistic regression with adjustment for BMI and SBP. For FOXO1A, two of its SNPs, rs2755209 and rs2755213, were associated with female longevity in a dominant and additive model (P less than 0.010.0.76 (0.62-0.92) 0.005 0.003 0.324/0.389 0.97 0.75 (0.60-0.94) 0.011 0.009 MAF, minor allele frequency; P, P-value from Hardy-Weinberg equilibrium test; OR (95% CI), odds ratio with 95% confidence interval; P, P-value from x2 HWT test; aP, adjusted P for BMI. Table 5. Ethnic effects on association of FOXO3A with longevity trait SNPs Han Chinese (this study) SNPs Japanese (12) SNPs German (13) MAF (case/control) P r2 MAF (case/control) P r2 MAF (case/control) P rs2253310 0.303/0.244 7.9 x | 1025 rs2802292 1 0.371/0.255, 0.0001 rs768023 1 0.44/0.382 0.007 rs2764264 0.87 0.347/0.248 0.0002 rs2802288 1 0.445/0.385 0.006 rs13217795 0.93 0.340/0.248 0.0006 rs2802290 1 0.444/0.387 0.01 rs9400239 0.93 0.366/0.295 rs2802292 0.302/0.240 2.9 x |1025 rs2764264 0.87 0.347/0.248 0.0002 rs768023 1 0.440/0382, 0007 rs13217795, and rs2802288 1 0.445/0.385 0.006 rs2802290 1 0.444/0.387 0.01 rs9400239 0.93 0.366/0.295 rs4946936 0.283/0.221 1.8 3 x | 1025 rs2764264 0.8 0.347/0.248 0.0002 rs9400239 0.87 0.366/0295, and rs13217795 086-0340248-00006 rs1268170 081-0397/0347 002 MAF, minor allele frequency; P, P-value from allelic association study; r2, pairwise linkage disequilibrium value in CHB from HapMap II between SNPs used in this and others (Japanese and German groups). DISCUSSION Replication study Three hundred fifty female centenarians and 350 geographically matched younger female individuals were referred to as Population 2 (Table 1) and used for replicating the finding that FOXO1A is associated with female longevity. We found that the MAFs of SNPs rs2755209 and rs2755213 from FOXO1A were significantly reduced in the centenarian group compared with control (adjusted P < 0.025 and <0.009, respectively) while that of SNP rs17630266 was not distinct between the centenarian and control groups (Table 4). These results suggest that FOXO1A is associated with female longevity in Population 2.

In this study, we demonstrated that FOXO1A is strongly associated with human female longevity and validated a previous finding that FOXO3A is associated with the longevity phenotype in the Han Chinese population. The association of FOXO1A with male longevity is not statistically significant in our study. It may be possible that the small male centenarian population in this study does not allow to unveil a weak association between FOXO1A genotypes and male longevity. But it appears true that a female longevity trait is more susceptible to the genetic variations of FOXO1A.