In this study, we demonstrated that FOXO1A is strongly associated with human female longevity and validated a pre- vious 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 centenar- ian 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 suscep- tible to the genetic variations of FOXO1A.

That women live longer than men has been recognized. However, studies on identifying the genetic causes for this phenomenon are very limited and preliminary (30). Pre- viously, Barbieri et al. (31) found that an exonic SNP repla- cing the amino acid Pro with Ala in peroxisome proliferators-activated receptor (PPAR) g-2 has different dis- tributions between long-lived males and controls (P ¼ 0.035), but not between female groups. Recently, it was found that an SNP (A/G)-308 in tumor necrosis factor has sex- dependent distributions, and allele A is specifically associated with male life expectancy (P ¼ 0.019) (32). In an animal model, it has been shown that the increased expression of dFOXO is associated only with female lifespan (21). Since D. melanogaster has only one form of FoxOs, it is impossible to know which form of FoxOs in mammals could be respon- sible for female longevity. Our study identified that FOXO1A is strongly associated with human female longevity.

Effects of ages and ethnicities on the MAF distributions of longevity-associated SNPs and haplotypes

The ages when blood samples were collected were classified into three groups in both male and female. Decreased MAFs of SNPs rs2755209 and rs2755213 with age were found for FOXO1A in females (Supplementary Material, Table S4). On the other hand, the MAFs of all three SNPs from FOXO3A were enriched with increased age in both genders (Supplementary Material, Table S4). This enrichment was also reported for the SNPs from FOXO3A in Japanese and German longevity studies; which are in the same haplotypes and associated with the longevity trait (Table 5).

(50–53). But when suffering from diseases such as diabetes, the aged female shows more increased insulin resistance and susceptibility to ischemic injury in the heart than the male (54). These suggest that gender-related human longevity may be associated with sexual dimorphism in insulin resist- ance. Although FOXO1A and FOXO3A are both direct down- stream molecules of AKT1 in insulin/insulin-like growth factor signaling pathways, their functions are not identical. For instance, mice without FOXO1A are embryonic lethal, but they are viable without FOXO3A, suggesting that FOXO1A is indispensable (35,55) and acts as a main factor mediating the insulin signaling pathway (56). Over-expression of dFOXO in D. melanogaster, an effect mimicking an impaired insulin signaling pathway, only protects females from paraquat, increasing their lifespan (21). These imply that FOXO1A plays a role in female longevity by regulating sex-dependent insulin sensitivity.

with human longevity has been debated (12,23,24). This is likely due, at least in part, to difficulties in collecting sufficient numbers of long-lived and well-controlled individuals from both genders who can be used for a genetic association study. To understand the possible reasons for the debate in the literature, we compared our study with others and found that, in addition to ethnicity, the sample sizes varied signifi- cantly. More importantly, we noted that none of the previous studies mentioned sexual dimorphism in the association of FoxOs with human longevity. The total numbers of centenar- ians were 761 in our original study and 350 in the replication, but only 122, 218 and 213 in the other three studies (12,23,24). It is possible that a small sample size, or improper sex combi- nation, produces insufficient statistical power, therefore redu- cing the probability of identifying FOXO1A associated with human female longevity. Here, we took full advantage of the large centenarian populations to determine the association of FOXO1A and FOXO3A with longevity within genders. We validated the association of FOXO3A with human longevity previously found in Japanese, German and Italian population- based studies (12–14) and replicated the finding that FOXO1A is not associated with male longevity (12). More importantly, we demonstrate that FOXO1A is associated with female long- evity, providing a new insight into how a genetic factor contributes to human longevity. The association of both FOXO1A and FOXO3A with the human longevity trait appears in additive and dominant models in Han Chinese. Interestingly, Han Chinese, Japanese, German and Italian share the same longevity-associated haplotypes of FOXO3A. This excludes the possible influences of population stratifica- tion on association studies.

In addition, FOXO1A is highly expressed in the female reproductive system, including ovaries and uterus, whereas FOXO3A is more ubiquitously expressed in vivo. Several studies suggest that FOXO1A plays a major role in the regu- lation of female decidualization (57–59). In women, delayed menopause is associated with age of death. For instance, death is often postponed for a few more decades if menopause occurs after the age of 50 (60). It is also known in D. melanogaster that over-expression of dFOXO reduces female fecundity and extends lifespan (21). Thus, it will be of great interest to know whether FOXO1A affects female longevity by regulating reproduction. But whether this is suf- ficient to explain why FOXO1A shows association with human female longevity needs to be determined.