Despite growing evidence for genetic determinants of long- evity, the identification of longevity-predisposing loci or genes remains very difficult because of strong environmental effects and the complexity of the longevity trait. Previously, genome- wide linkage analysis provided a link between the marker D4S1564 at chromosome 4 and the exceptional longevity phe- notype (7,8). However, this locus has been debated in a recent study (9). Several loci or genes, except for APOE, were found to be associated with longevity in some studies, but not repro- duced by others (10). A recent genome-wide association study in the Framingham population suggested that eight SNPs are strongly associated with the age of death. Among these eight, two SNPs, rs4943794 and rs10507486 located in the FOXO1A gene, present the strongest association tested in the generalized estimating equation (GEE) model (11). In addition, FOXO3A, the other FoxO member, has recently been associated with human longevity in Japanese, German and Southern Italian (12–14). FOXO1A and FOXO3A are critical downstream molecules of AKT1 and are inactivated by AKT1-mediated insulin signaling pathways that regulate the cell cycle, apoptosis, stress resistance and metabolism (15–18). It has been shown that the modification of FoxO in C. elegans (DAF-16) or in D. melanogaster (dFOXO) signifi- cantly affects their maximum lifespan (19–21), suggesting that FoxOs play an important role in the aging process. FOXO1A is recognized as a longevity factor (22). Although some controversies remain, these studies provide promising findings in the association of genetic factors with the human longevity trait (12,23,24).

RESULTS

Characterization of population

Two pairs of cases (centenarians) and controls (younger indi- viduals) were collected among the Han Chinese. One pair (Population 1) was from southern China whereas the other (Population 2) was from northern China. The mean ages of centenarians in Populations 1 and 2 were 102.3+ 0.14 and 100.8+ 0.19 years (mean + SEM). The male-to-female ratio was about 1:3 in centenarians and 1.8:1 in controls in Popu- lation 1. But in Population 2, only females were collected. Body mass index (BMI) and systolic and diastolic blood press- ures of both populations are presented in Table 1. The differ- ences between case and control groups for the listed parameters were evaluated by Student’s t-test. A P-value ,0.05 was considered statistically significant.

Single SNP association analysis

Six SNPs (rs17630266, rs2755209 and rs2755213 for FOXO1A; rs2253310, rs2802292 and rs4946936 for FOXO3A) were genotyped by direct sequencing in all selected individuals. No significant deviation from the Hardy– Weinberg equilibrium test was found for all six SNPs in both populations. In sex-combined centenarians and controls, except for SNP rs17630266, the minor allele frequencies (MAFs) of both rs2755209 and rs2755213 of FOXO1A were significantly lower in the centenarian group than in control.

In addition to genetic and environmental factors, also has strong effects on the human longevity phenotype. is well-known that females have a longer life than males (25). A number of studies have documented females account for a much larger percentage of the ian population (26). In the 1998 baseline survey of the Longitudinal Healthy Longevity Study (CLHLS), we that the ratio of males to females in centenarians is 1:4

Table 1. Characteristics of populations

Items Population 1 P Population 2 P Case (n ¼ 761) Control (n ¼ 1056) Case (n ¼ 350) Control (n ¼ 350) Gender (m/f) 183/578 682/374 0/350 0/350 Age (year) 102.3+0.14 47.1+0.22 ,0.001 100.8+0.19 41.4+0.48 ,0.001 BMI (kg/m2) 19.9+0.2 24.9+0.1 ,0.001 18.5+0.3 21.3+0.3 ,0.001 SBP (mmHg) 146+0.9 129+0.5 ,0.001 149+1.4 115+0.8 ,0.001 DBP (mmHg) 82+0.5 84+0.3 0.010 83+0.7 73+0.5 ,0.001

The data are presented as mean+SEM (standard error of the mean). P-values are calculated from t-test comparing case and control groups within population. Case, centenarian; Control: individuals from general population; m/f, male/female; BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure.

Table 2. Allelic association of FOXO1A and FOXO3A with longevity (sex-combined in Population 1)

SNP Allele (major/minor) Function MAF (case/control) P OR (95% CI) P bP PW (a ¼ 0.05) HWT rs17630266 G/T Intronic 0.392/0.372 0.50 1.09(0.95–1.25) 0.221 0.221 NC rs2755209 C/T Intronic 0.242/0.286 0.86 0.80(0.69–0.93) 4.6   1023 9.0 )x | 1023 0.83 rs2755213 C/T Intronic 0.352/0.420 0.53 0.75(0.65–0.86) 7.4   1025 3.0   1024 0.98 rs2253310 G/C Intronic 0.303/0.244 0.70 1.35(1.16–1.57) 7.9   1025 2.3   1024 0.97 rs2802292 T/G Intronic 0.302/0.240 0.74 1.36(1.18–1.60) 2.9   1025 1.5  1024 0.98 rs4946936 C/T 30-UTR 0.283/0.221 0.40 1.40(1.19–1.64) 1.8  3 x | 1025 1.1  Lx | 1024 0.99

MAF, minor allele frequency; P , P-value from Hardy–Weinberg equilibrium test; OR (95% CI), odds ratio with 95% confidence interval; P, P-value HWT calculated from x2 test; bP, corrected P by Bonferroni step-down method; PW, power to reach an alpha level of 0.05 with a given population size; NC, not calculated; 30-UTR, 3-untranslated region.

Table 3. Gender effects on allelic association of FOXO1A and FOXO3A with longevity trait in Population

SNP Male Female MAF (case/control) OR (95% CI) P bP MAF (case/control) OR (95% CI) P bP rs17630266 0.357/0.360 0.99(0.77–1.27) 0.914 0.91 0.402/0.392 1.04(0.86–1.27) 0.680 0.680 rs2755209 0.245/0.281 0.83(0.63–1.09) 0.166 0.33 0.243/0.294 0.77(0.62–0.95) 0.014 0.028 rs2755213 0.371/0.423 0.80(0.63–1.03) 0.073 0.22 0.348/0.413 0.76(0.62–0.92) 0.005 0.020 rs2253310 0.327/0.248 1.47(1.13–1.90) 0.003 0.014 0.296/0.235 1.37(1.10–1.71) 0.004 0.022 rs2802292 0.328/0.242 1.53(1.17–1.98) 0.001 0.006 0.295/0.236 1.36(1.09–1.69) 0.005 0.016 rs4946936 0.298/0.225 1.46(1.11–1.91) 0.004 0.017 0.280/0.212 1.44(1.15–1.82) 0.001 0.008

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