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. Flachsbart et al. (2009) and Li et al. (2009) reported signifi- cant findings in the populations composed of both genders. However, as seen in Table 1, 2, and 3, the differences in allele, genotype and haplotype frequencies in our cohorts were found to be most pronounced for males. The first study published on the association of SNPs in FOXO3A (Willcox et al. 2008) was conducted on an all male population and, moreover, five of six SNPs reported by Anselmi et al. (2009) were specific for males, whereas only one was specific for females, indicating a gender effect. Moreover, adding further support to a male-specific association in the case–control study we observe no associations in females even though the sample size is larger for females than

quencies between the younger controls and oldest old, we found nominally significantly differences between the oldest old males and male controls for five SNPs (rs12206094, rs13220810, rs2802292, rs3800231, and rs9486902) and one SNP (rs479744) was significantly different between centenarians and controls when analyzing both genders combined (see Table 1 and Supporting information Table S2). Of these six SNPs, all except rs12206094, rs13220210 and rs9486902 have been found to be associated with longevity at the allele level (after correction of multiple testing) in at least one previous study (Anselmi et al. 2009; Flachsbart et al. 2009; Li et al. 2009; Pawlikowska et al. 2009; Willcox et al. 2008). rs12206094 and rs948602 have to our knowledge not been reported before, whereas rs13220210 has been found to be nominally associated (Flachsbart et al. 2009). The associations of the SNPs point in the same direction as in previously published studies (the SNPs show an increase in frequency of the rare allele in old age, except rs13220810 where the most frequent allele was increased in the oldest old (see Supporting information Table S1)) supporting the findings. The associations with respect to allele frequencies did in our study, however, not hold after correction for multiple testing (see Supporting information Table S3). The fact that at the allelic level, our data does not hold for the correction of multiple testing might simply be a matter of sample size and hence lack of power to detect an association. However, our sample size is at least as large as the previously published studies. Moreover, as seen below, assum- ing a recessive model for the genotype frequency data generally gives the lowest PGGC values, indicating that the association effect is most pronounced for the rare homozygote individuals, and hence that the association effect might be ‘masked’ when analyzing the allele frequencies (that is the heterozygotes might

Flachsbart et al. (2009) and Li et al. (2009) reported signifi- cant findings in the populations composed of both genders. However, as seen in Table 1, 2, and 3, the differences in allele, genotype and haplotype frequencies in our cohorts were found to be most pronounced for males. The first study published on the association of SNPs in FOXO3A (Willcox et al. 2008) was conducted on an all male population and, moreover, five of six SNPs reported by Anselmi et al. (2009) were specific for males, whereas only one was specific for females, indicating a gender effect. Moreover, adding further support to a male-specific association in the case–control study we observe no associations in females even though the sample size is larger for females than for males (about 2.5-fold).

In addition to investigating differences in allele and genotype frequencies, we also investigated the possible haplotypes in FOXO3A. Again, we find that the effect is almost restricted to males (see Table 3 and Supporting information Table S4), and only for the TAC and CAC haplotypes of window 1 (composed by rs9486902, rs10499051, and rs12206094). The rs9486902 and rs12206094 were (as already described) found to be associ- ated at the genotype frequency level, so the observation that the haplotypes including these two SNPs are found to be associ- ated with longevity does not seem surprising.

Considering previous reporting (Flachsbart et al. 2009) of a most significant effect in centenarians, we analyzed the cente- narians separately. We were, however, not able to replicate this finding; that is, we did not find additional SNPs to be asso- ciated with longevity or an increased effect of the SNPs in the centenarians (compared to the controls) as when comparing the entire group of oldest old to the controls. The SNP found to be associated in both genders in the entire group of oldest old (rs7762395) was replicated in the centenarians; however, only one (rs2764264) of the eight SNPs found in the males of the entire group of oldest old was replicated in the centenari- ans. The reason for the latter is probably the low sample size of male centenarians (N = 30) and hence lack of power to detect associations.