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 two 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 hindered 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 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).

The age of the cases is crucial in genetic longevity research. In our study, the association of FOXO3A revealedThe text contains several errors and inconsistencies that need to be corrected. The processed text with the required corrections is given below.

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The centenarian sample answered matched for sex and geographic origin with healthy control individuals (553 younger controls ranged from 18 to 70 years; mean 51.2 years). The gender ratio of the sample was 83.6% females vs. 16.4% males. To test for population stratification, 57 microsatellites from the Applied Biosystems LMS-MD10 panel that were located on 6 different chromosomes (chromosomes 2, 9, 10, 11, 17, and 18) were tested in all cases and controls. Chi-squared (χ²) values were calculated from the allele counts. The obtained mean χ² of the G test statistics for the markers was 1.00, which reflected the good matching of the French sample collection (17). All German and French subjects gave informed, written consent before participation. The study was approved by the Ethics Committee of the Hospital Saint-Antoine in Paris.

Another difference between the two investigations may be the type of effect. The odds ratios (ORs) in the Japanese suggested an additive mechanism (12), whereas in the German sample, the data implicated a dominant-recessive effect. However, the large confidence intervals for the ORs in both studies imply some uncertainty. The future challenge is to investigate whether the observed discrepancies in the strength and kind of the FOXO3A effect can be verified and, if so, to clarify whether they are caused by study design, population- and/or gender-specific differences or some other factors.

There is growing evidence for an important role of the FOXO3A protein in healthy human aging. It has been shown to control insulin sensitivity and influence coronary heart disease, type 2 diabetes, and longevity. These functions are indicative of a master regulator in the IIS pathway, and allelic variation in the transcription factor FOXO3A may modulate a broad array of downstream targets that could exhibit larger effects on extending lifespan (12). The association findings in two independent populations of diverse origin confirm FOXO3A as a genetic susceptibility factor for human longevity.

Genotyping. Genotyping of the German and French samples was performed with Taqman SNP Genotyping Assays (Applied Biosystems) on an automated platform (23).

Statistical Analysis. Single-marker case-control analyses on allele and genotype frequency data were performed with χ² statistics by using the open-source software GENOMIZER (24) (www.ikmb.uni-kiel.de/software.html). P < 0.05 was considered significant. All analyzed SNPs had a minimal overall call rate of 98% and were tested for Hardy-Weinberg equilibrium (HWE) in controls before inclusion in the analyses (P > 0.05). The LD structure was determined from HapMap data HWE (CEU and JPT; www.hapmap.org) with the Haploview v4.0 program (http://www.broad.mit.edu/mpg/haploview) (25). The Haploview program was also used for selection of tagging SNPs based on the HapMap genotypes of Europeans with the pairwise r² > 0.8; P < 0.01 HWE and for the haplotype analysis.