The data of the 2 Dutch cohorts were also previously analyzed by applying a longitudinal design, including information on mortality and age-related phenotypes (15). Kuningas et al. (15) observed that carriers of a particular 4-SNP haplotype (GAGC from rs2802288, rs2883881, rs12200646, and rs13220810) had increased risks for all-cause and cardiovascular mortalities. The effects were very small, and the findings did not hold up to multiple testing (15). These 4 SNPs were also subjected to haplotype analysis in our sample collection. Although risk alleles or haplotypes for age-related diseases are expected to decrease in frequency in population strata of increasing age (16), the mortality haplotype identified in the Dutch was not significantly depleted in German centenarians compared with younger con-  trols (0.212 in centenarians versus 0.238 in controls; P 0.17).

rs2802292 rs2802288 Comparison OR 95% C.I. P O.R. 95% C.I. P Homozygote of minor vs. homozygote of major allele 2.75 1.51–5.02 0.0007 1.53 1.06–2.21 0.025 Heterozygote vs. homozygote of major allele 1.91 1.34–2.72 0.0003 1.54 1.16–2.04 0.0027 Carriers of minor vs. homozygote of major allele * * * 1.53 1.18–2.00 0.0016 Homozygote of minor allele vs. heterozygote * * * 1.01 0.71–1.42 0.970

Comparison

Discussion

et al. did not observe any association with FOXO3A in their cross-sectional comparisons. Our results highlight the relevance of centenarians for genetic longevity research, because they appear more valuable for such studies than octogenarians and nonagenarians. This finding might be attributed to the fact that centenarians represent the top percentiles of their respective birth cohort-specific age distributions and have outlived most of mw an  their peers by several decades. Only 4% of male and 5.6% of female 90-year-olds and 15–17% of 95-year-olds in Germany are likely to become 100 years themselves (Human Mortality Da- tabase, www.mortality.org), indicating that centenarians repre- sent a highly selected phenotype even among LLI. Because the genetic contribution to survival is strongest at very advanced ages (4), centenarians may be particularly enriched for beneficial variants in ‘‘longevity assurance genes’’ (16). Hence, it would seem that centenarians are the more suitable, but rarer, pheno- type for genetic longevity studies.

Recently, variation in FOXO3A was shown to be associated with longevity in a discovery cohort comprising 213 long-lived men of Japanese ancestry (95–106 years) and 402 younger probands (73–81 years) (12). Our study of 1,762 German centenarians, nonagenarians, and controls provides independent evidence that FOXO3A is a susceptibility gene for prolonged survival. As can be expected for a polygenic trait like longevity, the effect was rather modest (demonstrated by an OR of 1.42 for the top- ranking marker rs3800231; Table 2). However, because the association has been observed in 2 genetically diverse groups of European and Asian descent, our results render FOXO3A a modifier of general relevance that may play a role in many human populations. The analysis in the French centenarian sample generated a trend that supported the findings in the Japanese and German collections, but did not yield a statistically robust replication result. The validation could have been hin- dered by low levels of undetected population structure, but as demonstrated previously, the French sample shows no evidence of stratification (17). Another explanation might be the younger age of the French controls (18–70 years). This idea is supported by the fact that the estimated OR was larger when only controls aged 60–70 years were used for analysis. The resulting P CCA value still did not reach significance, which is possibly caused by the small number of available older controls (n Po, 137). The lack of reproducibility presents a well-known problem of case-control studies. To prove the validity of association results, confirmation in independent and larger samples is generally mandatory. However, this requirement may sometimes be difficult to achieve when polygenic traits, such as human longevity, are analyzed for which only modest or weak effects are predicted (5). This problem is aggravated in underpowered experiments where the sizes of appropriate samples are relatively small. The French centenarian-control collection used in this study is one of the largest to date and yet it had only an a priori power of 47% to confirm the association of the FOXO3A SNP rs768023 (assuming the same allele frequencies as in the German centenarian and control samples).

men was larger than that for the German collection of mixed gender. It is possible that this discrepancy could be attributed to an inaccuracy of estimation caused by chance (as reflected in the confidence intervals for the ORs). If the difference is actually real, the question arises as to which factors could have contrib- uted to it. First, it may be caused by some population-specific factors that result in a stronger effect in Japanese in general. Second, the association described by Willcox et al. (12) referred to males only. Men become long-lived less often, and males may depend more on genetic factors than females to attain excep- tional old age (19). It would therefore seem plausible that longevity variants are more enriched in men or impact more strongly on male survival. This may be the case for the Japanese men examined (12). However, our study also extended the association finding to females and indicates that genetic variation in FOXO3A is likely to contribute equally to the longevity phenotype in both genders. Third, the 2 investigations differed in the choice of controls. The selection of appropriate controls for genetic longevity research is a matter of concern (5, 14). we<7 Willcox et al. (12) used controls (mean age 77 years) whose birth cohorts were very close to those of the cases and who had already died before the age of 81. This so-called nested case-