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MAF, minor allele frequency; OR (95% CI), odds ratio with 95% confidence interval; P, P-value from x2 test; bP, corrected P by Bonferroni step-down method.

strong associations with longevity in both the dominant and additive models for males (P ¼ 0.005–0.001) and females (P ¼ 0.005–0.0002; Supplementary Material, Table S1).

The adjusted P for multiple comparison by the Bonferroni step- down procedure (bP) was from 9.0   1023 to 3   1024. Conversely, the MAFs of all SNPs of FOXO3A were much greater in the centenarian group than in control (bP ¼ 2.3 ) x 1024–1.1   1024). For the given sample size, the statistical power was 0.83–0.99 in the setting of an a level of 0.05 (Table 2).

Linkage disequilibrium and haplotype association analysis

Because the sex ratios were significantly different between the centenarian and control, we compared the frequencies of each SNP between case and control within genders. In males, none of the three SNPs from FOXO1A showed a stat- istic difference in MAF distributions between the case and control groups; whereas the three SNPs from FOXO3A pre- sented much higher MAFs in the centenarians than in the younger population (bP ¼ 0.017–0.006) with a power of 0.79–0.89 (Table 3). In comparison of the female groups, two SNPs of FOXO1A, rs2755209 and rs2755213, had reduced MAFs in the centenarian group versus the younger group (bP ¼ 0.028–0.02). The MAFs of all SNPs from FOXO3A were higher in centenarians than in the younger population in both genders (bP ¼ 0.022–0.008, Table 3).

Linkage disequilibrium (LD) and haplotype blocks  J IV 0.90) were defined and visualized by the solid spine of LD method (Supplementary Material, Fig. S1). The haplotypes with a frequency of (cid:4)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 distri- butions between case and control with statistical power .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 ¼ 0.009; Supplementary Material, Table S2).

Genotype association analysis

To test the inheritance models through which FOXO1A and FOXO3A are associated with longevity, we performed a gen- otype association test with dominant, recessive and additive models 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 domi- nant and additive model (P ¼ 0.01–0.005; Supplementary Material, Table S1). For FOXO3A, all three SNPs presented

We then tested the association of haplotypes from FOXO3A with male longevity, and found that, opposite to CGT, GTC had a reduced frequency in centenarians compared with con- trols. Statistical powers for significant haplotype association in the x2-test were .0.99. The statistical differences remained in the permutation test (Supplementary Material, Table S3).

Table 4. Validation of allelic association of FOXO1A with female longevity in Chinese populations

SNP Population 1 Population 2 MAF (case/control) P OR (95% CI) P aP MAF (case/control) P OR (95% CI) P aP HWT HWT rs17630266 0.402/0.392 0.81 1.04(0.86–1.27) 0.680 0.838 0.407/0.376 0.20 1.14(0.91–1.42) 0.240 0.203 rs2755209 0.243/0.294 0.32 0.77(0.62–0.95) 0.014 0.011 0.220/0.268 0.80 0.77(0.60–0.99) 0.039 0.025 rs2755213 0.348/0.413 0.84 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 0.440/0.382 0.007 rs13217795 0.93 0.340/0.248 0.0006 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 0.366/0.295 rs13217795 0.86 0.340/0.248 0.0006 rs1268170 0.81 0.397/0.347 0.02

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 geographi- cally 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 ¼ 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 longev- ity in Population 2.