Recently, Willcox and colleagues demonstrated an associ- ation between exceptional longevity and the FOXO3A gene in an ethnic Japanese population in Hawaii, focusing on male individuals only.22 Although there was no population strat- ification effect observed in this study, it is a possible limita- tion in most studies. Stratification is the result of imbalance of ethnic background between cases and controls. It is usu- ally modest when the recruitment is consistent among cases and controls, and for populations that did not experience re- cent immigration. In the present study, we extrapolated the FOXO3A locus from our genome-wide association data on a part of the SICS. Particularly, we genotyped (with BeadChip 300k, Illumina) 281 SIC male nonagenarians and 195 SIC male controls. It was encouraging to report the association between rs2802292 at the FOXO3A locus and the longevity phenotype was convincingly and independently confirmed in our SICS homozygous minor versus homozygous major alleles (p   0.0019; OR  2.58; CI 95%, 1.42–4.70). Moreover, to avoid the possible drawback of population structure, we demonstrated a small or null confounding effect of popula- tion structure on our association results, thus excluding type I errors (Fig. 1, Supplemental Table 2). Our aim was to repli- cate rs2802292 in male longevity and to explore the FOXO3A locus in males and females to generate eventual new candi- date SNPs for longevity and to clarify the gender interaction on the FOXO3A locus and longevity. Replication efforts re- quire adequate sample size and our male population was »>9 sufficient to achieve 90% power to detect an allele with 40% allele frequency and a factor of 1.5 effect, as previously re- ported in Willcox paper for rs2802292.22

This is the first convincing replication of an association with longevity after the APOE association.5 As stressed by National Cancer Institute–National Human Genome Re- search Institute (NCI-NHGRI) Working Group on Replica- tion in Association Studies,39 validation of the postulated gene-investigated phenotype association in a population dif- ferent from that of the previous study is of great value.

It is also established that mitochondrial Sirtuin 3 interacts with, and under specific cellular conditions regulates, the ac- tivity of FOXO3A. Additionally, SIRT3 regulates a series of essential intracellular processes that defend the cell against multiple types of cellular damage, including oxidative dam- age.40 A rise in the level of reactive oxygen species (ROS) has two important effects: It can damage proteins, lipids, and DNA, leading to cell death or it can trigger the activation of specific physiologic signaling pathway.41 Moreover, a GH- IGF1 pathway has been implicated in determination of longevity in a variety of species.18,19,42 Genetic variants that are either positively or negatively selected as population ages impact on survival (demographic selection). In this regard, the FOXO3A gene SNP rs2802292 should have a protective role.

These observations imply that the FOXO3A gene SNP rs2802292, or SNPs in LD, have a protective role by par- tially increasing the ability of other proteins to activate FOXO3A, or by increasing FOXO3A activity on down- stream targets. Resequencing of the FOXO3A locus will clarify the nature of the genetic variation that is tracked by rs2802292. It is possible that the variation does not need to affect coding genes, because coding genes are less than one- third of the evolutionary conserved genome.38 Further- more, future population studies on diseases of aging will clarify if FOXO3A associates across diseases, like the longevity gene APOE and Alzheimer and cardiovascular diseases. From the data that we obtained in our population,

Meta-analysis represents a well-established method for summarizing results and drawing conclusions from differ- ent studies for a set of common hypotheses. Therefore, we applied this approach to combine our association results for the SNP rs2802292 with the findings coming from Willcox et al. study and to achieve more statistical power. The null-sig- nificance of the Q-statistics29 (p  0.88) and the inconsistency

the association is strictly linked with the male gender. Tak- ing in consideration this new evidence, we would suggest a gender-specific analysis for the longevity phenotype. To conclude, the discovery and replication of a convincing as- sociation of FOXO3A locus contributes to the hypothesis that the impact of the IGF-I–insulin pathway on longevity is a property that has been evolutionarily conserved throughout the animal kingdom.

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