For both genders combined, haplotypes in the following windows were found to be nominally significant: window 1, 2, 3 and 4 (composed by SNPs rs9486902, rs10499051, rs12006094;

Table 3 Case-control association analysis of FOXO3A haplotypes with longevity. (A) males and females combined and (B) for males separately

Window SNPs in window Haplotype Uncorrected PCCH Corrected PCCH (A) Females and males Oldest old (N = 1089), N (controls) = 736 WIN1 rs9486902|rs10499051|rs12206094 TAC 0.0214 0.6068 WIN2 rs10499051|rs12206094|rs2802292 ACG 0.0421 0.9540 WIN3 rs12206094|rs2802292|rs13220810 CTC 0.0448 0.3646 WIN4 rs2802292|rs13220810|rs2764264 TCT 0.0390 0.3756 (B) Males Oldest old (N = 313), N (controls) = 371 WIN1 rs9486902|rs10499051|rs12206094 CAT 0.0478 0.4654 TAC 0.0052 0.0369 CAC 0.0008 0.0109 WIN2 rs10499051|rs12206094|rs2802292 ATG 0.0093 0.1918 ACG 0.0171 0.2687 ACT 0.0079 0.0629 WIN3 rs12206094|rs2802292|rs13220810 CTC 0.0125 0.1499 TGT 0.0056 0.1089 WIN4 rs2802292|rs13220810|rs2764264 GTC 0.0467 0.5644 TCT 0.0104 0.1189 WIN5 rs13220810|rs2764264|rs7762395 CTG 0.0226 0.2470 WIN13 rs3800231|rs3800232|rs479744 GGC 0.0357 0.3546

Only PCCHs (P-values obtained from haplotype based case-control comparison) being nominal significant (P < 0.05) are included in the table; data passing nominal significance are listed in columns named Uncorrected PCCH, whereas values corrected for multiple testing are listed in column named Corrected PCCH. Values passing correction by multiple testing are in bold. N, number of individuals in group.

genders combined; rs10499051 (GG) HR = 0.360, P = 0.023, 95%CI = (0.149-0.869) for females only; rs7762395 (AA) HR = 2.065, P = 0.009, 95%CI = (1.195-3.569) for males only; and rs9486902 (TT) HR = 1.739, P = 0.041; 95% CI = (1.022- 2.951) for males only. When correcting for multiple testing by Bonferroni correction (P < 0.05/2*15 SNPs = 0.00166) it was, however, evident that none of the results remained significant.

Case–control analysis of the centenarian group separately

One hundred and forty three of the 1089 1905 cohort reached 100 years of age. Because the associations of SNPs with longevity have previously been reported to be significant for centenarians (Flachsbart et al. 2009), we ducted the association studies for the centenarian group rately.

Discussion

At the nominal significance level, most of the SNPs and haplo- types found to be nominally significant for the entire group of oldest old were also found to be significant for the centenarians (the PCCAs, PCCGs, and PCCHs for the centenarian group are listed in Supporting information Tables S2 and S4). When cor- recting for multiple testing (see Supporting information Table S3), only the PCCGs for rs772395 (both genders com- bined) and rs2764264 (males only) were replicated in the cente- narians (P-values (corrected) = 0.016 and 0.036, respectively), whereas rs479744 was found to be significant for both genders combined (P-value (corrected) = 0.024) not in males only, as was found for the entire group of oldest old.

Numerous genes and variations have up until now been investi- gated for their contribution to human longevity, often demon- strating difficulties in replicating the initial findings, the only exception being the APOE gene (Christensen et al. 2006). How- ever, more and more data points to the FOXO3A gene as the next convincing longevity candidate gene.

In this study, we investigated the possible association of varia- tion in the FOXO3A gene with human longevity by covering the known common genetic variation of FOXO3A using tagging SNPs and analyzing the frequency data both by a case–control and by a longitudinal approach.

First, we conducted case–control analyses and found [as seen in previous studies (Anselmi et al. 2009; Flachsbart et al. 2009; Li et al. 2009; Pawlikowska et al. 2009; Willcox et al. 2008)] that the frequencies of the rare alleles and rare genotypes tended to be increased in the oldest old when compared to the younger controls (see Supporting information Table S1). One exception was the rs13220810 where the most frequent allele and most frequent genotype were increased in the oldest old. This was also observed by Flachsbart et al. (2009). Moreover, as

FOXO3A SNPs and longevity – Longitudinal study of an extinct cohort of oldest old

In addition to the case-control setup, we analyzed the follow-up survival data for oldest old (the results are listed in Supporting information Table S5). When conducting Cox regression, rs10499051, rs7762395 and rs9486902 were found to be nom- inally associated with longevity: rs10499051 (GG) Hazard rate (HR) = 0.496, P = 0.049, 95%CI = (0.247-0.997) for both

reported by Flachsbart et al., we found that the allele and geno- type frequencies for the nonagenarians in our cohort were gen- erally in between the frequencies for the younger controls and for the centenarians, supporting the reported increase in allele and genotype frequencies with age.

(rs13217795, rs2764264, rs479744, and rs9400239) have previously been reported to be associated (after correction for multiple testing) in at least one study (Flachsbart et al. 2009; Willcox et al. 2008), whereas three SNPs (rs13220810, 7762395, rs9486902) have been shown to be nominally signif- icant (Flachsbart et al. 2009; Pawlikowska et al. 2009). For all these SNPs (except rs13220810), the recessive model shows the lowest PGGC values, indicating that the association effects may be most pronounced for the rare homozygotes, possibly giving these individuals a survival advantage. The rs13220810, on the other hand, shows significance only when applying the dominant model, indicating that the positive effect of this SNP is most pronounced for individuals holding the common allele.