In genetic longevity research, the age of the cases is of utmost importance. In our study, the association of FOXO3A revealed stronger effects in the centenarians than in the nonagenarians or the whole LLI sample (Tables 1–3). The marker rs2802288 showed a continuous increase in the frequency of the minor allele with age. A remarkable rise was observed from the age group 100–104 years (44.1%) to that of 105–110 years (52.4%) (Table 6). However, it should be taken into account that there were only 21 individuals who were 105 years and older. It has recently been shown by computer simulation that samples of nonagenarians need to be 5 times larger than those of cente- narians to achieve comparable power and that the performance of association studies is drastically reduced when nonagenarians are considered as cases (18). It can be expected that the power is even more decreased when octogenarians are used, as was done in the Dutch study (15), which may explain why Kuningas

Table 6. Minor allele frequency distribution of rs2802288 in Germans by age groups

Age at Minor allele recruitment, frequency of years n rs2802288 60–75 731 0.385 95–99 631 0.402 100–104 362 0.441 105–110 21 0.524

SNPrs2802288isinperfectLDwithWillcoxetal.’s(12)rs2802292(according CEU HapMap data),

control design avoids or minimizes a number of potential pitfalls that may arise when controls are drawn from much more recent birth cohorts than the LLI and are still alive at the time of recruitment (13, 14). The latter strategy was applied for the German association study described here.

harvard.edu/purcell/plink), based on 290 randomly chosen, genomewide Hospital Schleswig–Holstein, Campus Kiel and local data protection

Hospital Schleswig–Holstein, Campus Kiel and local data protection authorities. The French sample consisted of 535 centenarians (mean age: 103.8 years) from different regions throughout France (Iˆle-de-France, Northeast, North- west,Southeast,Southwest)(22).Thecentenarianswerematchedforsexand 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 lo- catedon6differentchromosomes(chromosomes2,9,10,11,17,and18)were tested in all cases and controls. (cid:1)2 values were calculated from the allele counts.Theobtainedmean(cid:1)2oftheGteststatisticsforthemarkerswas1.00, which reflected the good matching of the French sample collection (17). All German and French subjects gave informed, written consent before partici- pation. The study was approved by the Ethics Committee of the Hospital Saint-Antoine in Paris.

Another difference between the 2 investigations may be the type of the effect. The 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 2 independent popu- lations of diverse origin confirm FOXO3A as a genetic suscep- tibility 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 geno- type frequency data were performed with (cid:1)2 statistics by using the open- sourcesoftwareGENOMIZER(24)(www.ikmb.uni-kiel.de/software.html).P(cid:5) 0.05 was considered significant. All analyzed SNPs had a minimal overall call rate of 98% and were tested for HWE in controls before inclusion in the analyses (P r>C 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 Europeanswiththepairwisetaggingoption(pairwiser2(cid:6)(cid:1)0.8;P (cid:6)0.01) HWE and for the haplotype analysis.

Materials and Methods