In summary, we demonstrate in this study that FOXO1A is associated with female longevity while FOXO3A is associated with longevity in both genders. The association of both FOXO1A and FOXO3A with the human longevity trait is inherited in additive or dominant fashions. Han Chinese shares the same longevity-associated haplotypes of FOXO3A with Japanese and German. Although the finding that FOXO1A is not associated with male longevity has been repli- cated in Han Chinese and Japanese populations, the numbers of male centenarians are relatively small in both studies. Therefore, it needs to be cautious. The association of FOXO1A and FOXO3A with human longevity needs to be validated in more ethnic groups and in larger populations.

FOXO1A and FOXO3A, members of the forkhead tran- scription factors of the FoxO family, serve as the direct down- stream signaling molecules of AKT1 in insulin/insulin-like growth factor signaling pathways. In vivo, FOXO1A and FOXO3A regulate the cell cycle and growth, apoptosis, DNA damage responses and angiogenesis (33–36). Malfunc- tions of FOXO1A or FOXO3A are involved in various cancers, insulin resistance, altered immune responses and organ damage (15,22,37–40). In the cardiovascular system, for example, FOXO1A and/or FOXO3A are/is important for the onset of diabetic cardiomyopathy (41,42), cardiac hyper- trophy (43,44) and ischemic heart disease (45,46). It is likely that FOXO1A and FOXO3A affect longevity through multiple pathways, such as insulin resistance, stress responses or proneness to disease. Earlier studies have provided several lines of evidence that both FOXO1A and FOXO3A are associ- ated with HbA1c level and fasting plasma insulin (12), (47,48), suggesting that their contributions to human longevity may be due to balancing insulin sensitivity and insulin resist- ance through insulin/insulin-like growth factor signaling path- ways. The question remains why FOXO1A is more closely associated with human female longevity.

MATERIALS AND METHODS

Study subjects

We performed the baseline survey of the CLHLS reported pre- viously (61). In the Survey, we interviewed 9093 oldest-olds aged 80–116 with a questionnaire containing 404 questions and physical tests throughout 85% of the regions of China. Among these 9093, 8441 were Han Chinese. In this study, a total of 761 centenarians (long-lived group) and 1056 unre- lated younger individuals (control group) from southern China were selected and used as Population 1 for initial screening of all six SNPs. We also took 350 female centenar- ians and 350 younger individuals from northern China for the necessary replication of associations found in the initial

It has been reported that insulin sensitivity is highly sex- differentiated at different developmental stages or under different stresses in both humans and animal models (49,50). In female rats, for example, a high-fat or high-sugar diet does not induce insulin-resistance as seen in males, indicating that females have a gender-dependent protective effect

were subjected to an association test with longevity. The per- mutation P-value was obtained by simulating 100 000 times in haplotype association analysis (64).

screening. All participants were Han Chinese. Finger-prick blood samples from centenarians were spotted onto S&S no. 903 filter paper (Schleicher & Schuell, Germany) and stored at 48C after the spots were completely dried. Two to three milliliters of blood were obtained from each younger individual. Written informed consent was obtained from all participants or their representative family members in cases of centenarians who were incapable of signing. BMI was cal- culated based on the formula (body weight/height2). The basic characteristics of the studied populations are presented in Table 1. The study protocol was approved by the Institutional Review Board, Institute of Molecular Medicine at Peking Uni- versity. The study conformed to the principles outlined in the Declaration of Helsinki.

The two-tailed P-values, odds ratios and 95% confidence intervals are presented for all association tests. Statistical power was calculated with the sampsi command in STATA (StataCorp LP) under a given sample size and significance level (a ¼ 0.05).

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG online.



Genotyping of SNPs

We thank all participants and their families for their commit- ment and enthusiastic help in the Chinese Longitudinal Healthy Longevity Survey.

Human genomic DNA was isolated from a 5-mm diameter punch-out from each blood spot or EDTA-anticoagulated blood using the proteinase K methods described previously (62). Based on the HapMap (CHBþJPT), the tagging SNPs rs17630266, rs2755209 and rs2755213 were selected for FOXO1A, and rs2253310, rs2802292 and rs4946936 for FOXO3A. DNA fragments of 200–350 bp containing SNPs were amplified by PCR from 10 ng of genomic DNA from each participant, with the primers listed below. The amplified DNA fragments were purified and used for genotyping by direct- sequencing with a BigDye v1.1 kit and running on ABI 3130XL. Based on the GenBank numbers NT_024524 for FOXO1A and NT_025741 for FOXO3A, the pairs of PCR primers for amplifi- cation of rs17630266, rs2755209 and rs2755213 for FOXO1A as well as rs2253310, rs2802292 and rs4946936 for FOXO3A were 50-GGTGATGGCAGTGACTGTCTC-30/50-GTGGGTACAG CAGACAAGGCT-30; 50-GATCAGCTGGCATTCCCAG -30/50-CAGTGCCACTGTGTCTCTG-30; 50-TGTATATTC AAGGTATGTTCC-30/50-CTTAGTAAACAGACTATGTAT CC-30; 50-GAGCTTGCTTTGGAGATGCA-30/50-CCCAGT CACTCACATAGTCCT-30; 50-CTGAGGCTAACAGCT GGGTCT-30/50-CACTGGCTGCCTGACACCTAT-30; and 50-GGGTCCTGAGAACTTCTGAGT-30/50-GACATTCTGT AAGACATTCTGCCT-30, respectively.

Conflict of Interest statement. None declared.

FUNDING