Author contributions: F.F., H.v.E.-E., S.N., S.S., and A.N. designed research; F.F., R.K., H.B., andS.N.performedresearch;F.F.,A.C.,R.K.,H.B.,andA.N.analyzeddata;andF.F.,A.C.,and A.N. wrote the paper.

contributions: F.F., H.v.E.-E., S.N., S.S., and A.N. designed research; F.F., R.K., H.B., wrote the paper. authors declare no conflict of interest. article is a PNAS Direct Submission. and A.N. contributed equally to this work. whom correspondence should be addressed. E-mail: s.schreiber@mucosa.de. article contains supporting information online at www.pnas.org/cgi/content/full/ 2009 by The National Academy of Sciences of the USA

The human forkhead box O3A gene (FOXO3A) is one of the homologues of daf-16 in C. elegans. The DAF-16 protein is a transcription factor and an evolutionarily conserved key regu- lator of the insulin–IGF1 signaling (IIS) pathway that influences metabolism and lifespan in model organisms (9–11). These aspects also render FOXO3A a very likely candidate for genetic longevity studies in humans. Recently, Willcox et al. (12) de- scribed 3 SNPs in the FOXO3A gene that were statistically significantly associated with longevity and different aging phe-

© 2009 by The National Academy of Sciences of the USA

Table 1. Association statistics for 16 FOXO3A SNPs in German LLI (subset A)

Min AF controls, Min AF LLI, No. dbSNP ID n   731 n   1,031 P P OR 95% C.I. CCA CCG 1 rs2274776 0.490 0.485 0.747 0.807 0.98 0.85–1.12 2 rs1571631 0.465 0.463 0.893 0.987 0.99 0.87–1.13 3 rs6911407 0.378 0.415 0.030 0.101 1.16 1.01–1.34 4 rs768023 0.382 0.415 0.051 0.134 1.15 1.00–1.32 5 rs2802288 0.385 0.417 0.058 0.146 1.14 1.00–1.31 6 rs2883881 0.099 0.084 0.129 0.019 0.83 0.66–1.05 7 rs12200646 0.118 0.141 0.050 0.141 1.22 1.00–1.50 8 rs2802290 0.387 0.417 0.081 0.185 1.13 0.99–1.30 9 rs13220810 0.263 0.248 0.308 0.125 0.92 0.79–1.08 10 rs7762395 0.153 0.175 0.090 0.191 1.17 0.98–1.41 11 rs9400239 0.295 0.331 0.025 0.033 1.18 1.02–1.37 12 rs3800231 0.287 0.327 0.011 0.025 1.21 1.04–1.40 13 rs1268170 0.347 0.377 0.073 0.171 1.14 0.99–1.31 14 rs473268 0.337 0.366 0.075 0.196 1.14 0.99–1.31 15 rs479744 0.199 0.229 0.033 0.082 1.20 1.01–1.41 16 rs519007 0.186 0.183 0.816 0.796 0.98 0.82–1.16

LLI, entire sample; 95–110 years. Min AF, minor allele frequency; P , P value obtained from an allele-based CCA comparison, using a (cid:1)2 test with 1 df; P , P value obtained from a genotype-based case-control CCG

associations, we looked at the SNP allele frequencies of the ain  nonagenarians separately (n 643; aged 95–99 years; mean age 96.5 years). As expected, their frequencies were intermediate between those of the controls and the whole LLI sample (Table 3).

were spaced evenly across the FOXO3A gene region and located in different haplotype blocks (Fig. 1). No evidence for an association was detected between any of the tested SNPs and the longevity phenotype (Table 4). SNP rs768023 yielded the lowest P value (0.097) and had an allele frequency distribution that CCA was close to that in Germans. When we repeated the analysis with only the older French controls (137 individuals aged 60–70 years), whose age range was comparable with that of the Germans, a similar P value for rs768023 (0.094) and a larger CCA estimated OR (1.26) were observed.

In the following we concentrated our analyses on the cente- narians. When we analyzed the 16 SNPs by logistic regression, incorporating APOE status and gender with possible interac- tions, we obtained only negligible differences to the unadjusted results. There was no significant interaction between any of the SNPs and either APOE status or gender in the impact on longevity.

Recently, Willcox et al. (12) reported an association of 3 FOXO3A SNPs with longevity in Japanese–American men from the island of Oahu (Hawaii). Unfortunately, there is no overlap between the 16 polymorphisms tested here and the 3 investigated in the Hawaii sample as the genotyping in the German and French samples had been conducted before publication of the Willcox study. However, based on LD data from HapMap individuals of European ancestry (CEU), the SNPs described by

Three of the SNPs that were associated in the German centenarian sample (rs768023, rs7762395, and rs3800231) were subsequently investigated for replication in an independent collection of 535 French centenarians (mean age: 103.8 years) and 553 younger controls (aged 18–70 years). The 3 SNPs included rs3800231 (the top-ranking marker in Germans); they

Table 2. Association statistics for 16 FOXO3A SNPs in German centenarians (subset B)

Min AF controls, Min AF centenarians, No. dbSNP ID n   731 n   388 P P OR 95% C.I. CCA CCG 1 rs2274776 0.490 0.477 0.536 0.373 0.95 0.79–1.13 2 rs1571631 0.465 0.461 0.872 0.543 0.99 0.83–1.17 3 rs6911407 0.378 0.438 0.006 0.012 1.28 1.07–1.53 4 rs768023 0.382 0.440 0.007 0.010 1.27 1.07–1.52 5 rs2802288 0.385 0.445 0.006 0.007 1.28 1.07–1.53 6 rs2883881 0.099 0.078 0.100 0.010 0.77 0.56–1.05 7 rs12200646 0.118 0.146 0.064 0.157 1.28 0.99–1.65 8 rs2802290 0.387 0.444 0.010 0.008 1.26 1.06–1.51 9 rs13220810 0.263 0.218 0.020 0.053 0.78 0.63–0.96 10 rs7762395 0.153 0.199 0.005 0.012 1.38 1.10–1.73 11 rs9400239 0.295 0.366 0.0007 0.0009 1.38 1.14–1.66 12 rs3800231 0.287 0.362 0.0002 0.0003 1.42 1.18–1.70 13 rs1268170 0.347 0.397 0.020 0.051 1.24 1.03–1.48 14 rs473268 0.337 0.380 0.044 0.078 1.21 1.01–1.45 15 rs479744 0.199 0.258 0.001 0.002 1.40 1.14–1.73 16 rs519007 0.186 0.176 0.550 0.799 0.93 0.74–1.17

For abbreviations see legend to Table 1.