Table 5). A comparison of the two SNPs reveals a comparable increase in their minor allele frequencies in controls relative to LLI (2.9% in Japanese (Kojima et al., 2004) and 4.4% in Chinese (Li et al., 2009). In Japanese, this difference is not significant, which may be because of the relative small sample size used in that study (122 centenarians and 122 controls). Also the other two FOXO1 SNPs of Li et al. (2009) show similar allele frequen- cies in both Chinese and Japanese (Table 5), which raises the intriguing possibility of a continent-specific effect. This observa- tion is underscored by the notable differences in LD structure and allele⁄haplotype distribution between European and Asian populations [Tables 4 and 5, Fig. 2 and HapMap data (Consor- tium 2003)]. Therefore, although our study did not show any evidence for an association of FOXO1 variants with human lon- gevity in Germans and Italians, this does not exclude an effect that could be restricted to Chinese or Asians. Longevity in differ- ent populations is likely to be influenced by varying sets of inter- acting genetic and environmental factors (e.g. diet) (Caliebe et al., 2010). To confirm the Asian- and female-specific longevity association of FOXO1, replications with more exten- sive DNA collections of, for instance, Japanese origin would be helpful.

FOXO1 110.9 kb 3′ 5′ 9 4 5 6 9 4 0 7 0 1 3 1 2 4 8 7 9 9 2 4 7 s s s r r r (B) Haplotype min AF cases min AF controls P value C G A 0.417 0.411 0.652 C G G 0.274 0.261 0.294 T C A 0.211 0.223 0.330 T G A 0.097 0.104 0.378

Fig. 1 (A) Gene structure of FOXO1. (B) Results of the haplotype analysis in German long-lived individuals and controls. Single-nucleotide polymorphisms are based on the study of Kuningas et al. (2007).minAF, minor allele frequency.

and most comprehensive one to date to assess the genetic con- tribution of FOXO1 to the phenotype (1886 German and 382 Italian females). Our overall result suggests that polymorphisms in none of the genes have a significant influence on a person’s ability to reach old age.

For FOXO1, several longevity association studies have previ- ously been performed, albeit with contradictory findings (Bona- fe et al., 2003; Kojima et al., 2004; Li et al., 2009). Bonafe et al. (2003) described a negative result in Italians that is – though only a single SNP was tested – consistent with ours. Recently, a study of Han Chinese found two SNPs as well as two haplotypes in this gene to be associated with longevity in women (Li et al., 2009). The results of the single-point analysis were also replicated in an additional sample of Chinese (Li et al., 2009). However, two previous investigations of Asian populations had revealed no association (Kojima et al., 2004; Willcox et al., 2008). One of the studies that examined only male individuals of Japanese des- cent (Willcox et al., 2008) is not informative with regard to a potential association in females. Another report analyzing Japa- nese of both genders tested three SNPs (Kojima et al., 2004), which were not haplotype-tagging and thus did not cover the allelic variation in FOXO1 comprehensively. Of note, one of the SNPs, rs2297627, is in strong LD with one of the significantly associated markers described in Han Chinese (rs2755209,

Polymorphisms in FOXO3A have convincingly and repeatedly been associated in numerous populations with the ability to sur- vive into old age (Willcox et al., 2008; Anselmi et al., 2009; Flachsbart et al., 2009; Li et al., 2009; Pawlikowska et al., 2009; Soerensen et al., 2010). Although here we used a larger sample than the one genotyped for the FOXO3A analysis (Flachsbart et al., 2009), our results did not ascertain an association of FOXO1, FOXO4, or FOXO6 with longevity in Germans. The members of the FOXO gene family share some characteristics (more than 60% sequence identity of the human FOXO domain (Anderson et al., 1998), regulation via the IIS pathway and a seemingly functional redundancy in vitro (Coffer & Burgering, 2004)) and yet their physiological roles are thought to be diverse (Wang et al., 2009), a finding that is supported by different expression patterns (Furuyama et al., 2000; Biggs et al., 2001). This functional diversification is also illustrated by the disruption of the FOXO genes (Hosaka et al., 2004): while a homozygous knock-out of FOXO1 is embryonically lethal, FOXO3A)⁄) and FOXO4)⁄) animals are viable and initially develop normal.

Table 3 Association statistics of FOXO1 single-nucleotide polymorphisms (SNPs) in female Italian individuals and in the pooled sample of Germans and Italians

minAF LLI minAF controls P allele P geno OR (95% C.I.) SICS LLI = 166 rs2755209 0.404 0.391 0.728 0.379 1.05 (0.79–1.41) Controls = 216 rs2755213 0.124 0.118 0.795 NA 1.06 (0.68–1.64) Germans + SICS LLI = 1258 rs2755209 0.373 0.379 0.702 0.601 0.98 (0.86–1.10) Controls = 1010 rs2755213 0.092 0.099 0.445 0.271 0.92 (0.76–1.13)

SICS, South Italian Centenarian Study; minAF, minor allele frequency; LLI, long-lived individuals; n, number of individuals; P allele, P value obtained from allele-based case–control comparison using a chi square-test with 1 degree of freedom (df); P geno, P value obtained from genotype-based case–control comparison using a chi square-test with 2 df; OR, estimated odds ratio for minor allele (based on whole sample); CI, 95% confidence interval of OR; NA, genotype-based case–control comparison was not conducted when there were fewer than five observations.

Experimental procedures

Table 4 Comparison of FOXO1 haplotype association results in German and Chinese females

Germans Chinese(cid:2) Freq. Freq. P Freq. Freq. P Haplotype† cases controls value cases controls value ATG§ 0.608 0.614 0.768 0.230 0.292 0.003 CTG 0.292 0.287 0.811 0.116 0.127 0.550 CCG 0.060 0.061 0.901 0.248 0.192 0.006 CCT 0.031 0.027 0.581 0.392 0.380 0.604 CTT 0.005 0.007 0.478 0.008 0.004 0.380

Study participants