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The population-based Leiden 85-plus study (van Heemst et al., 2005) also found a FOXO3A haplotype, but no single SNPs, associated with an increase in mortality (Kuningas et al., 2007) in Caucasians. The FOXO3A haplotype identified contains some of the SNPs we found nominally associated with longevity in SOF, but resides in a different part of the gene from the two FOXO3A SNPs we found associated with longevity in our three- cohort meta-analysis. Taken together, these results suggest that 5 independent cohorts may be detecting an association between FOXO3A variants and longevity. A combined analysis in which all cohorts genotype the same SNPs will be useful to confirm whether these findings are true replications of the same effect, and to identify the region of the gene with the strongest

Other insulin/IGF1-pathway genes implicated in human lon- gevity include FOXO1A and IGF1R. A genome-wide association scan in the Framingham Study showed association between age at death and two FOXO1A SNPs (Lunetta et al., 2007), which are in complete LD with FOXO1A rs1334241, the SNP we found to be nominally associated with longevity in SOF. However, this

 

Fig. 1 Graphic of insulin/IGF1 signaling pathway showing proteins encoded by scanned genes.

area, it may not be possible to discern which is causative from association studies in Caucasians alone; association studies in other populations, locus re-sequencing and functional studies are needed.

finding did not replicate in CHS or AJC. In the AJC study, rare nonsynonymous mutations of IGF1R were significantly more common in female centenarians (Suh et al., 2008). Female cen- tenarians carrying those mutations had lower IGF1R levels and decreased IGF signaling compared with other female centenari- ans (Suh et al., 2008). In a study of long-lived Italians, those car- rying a common variant allele at the IGF1R locus had lower plasma IGF1 levels than those without the allele; this allele was over-represented in long-lived Caucasians (Bonafe et al., 2003). We genotyped this variant but did not replicate the association with lifespan in the SOF cohort.

The AKT1 SNP associated with lifespan is not in high LD with any other HapMap variants; evaluation of eight unlinked vari- ants in the nearby area, including five amino-acid coding poly- morphisms, did not reveal any further associations with lifespan. As AKT1 rs3803304 resides 70 bp away from an intron–exon boundary, and in an area of high-predicted regulatory potential (King et al., 2005), which potentially encodes a noncoding regu- latory RNA (Kapranov et al., 2007), it may be a candidate SNP for influencing RNA splicing or other regulatory mechanisms. In vitro functional studies will be needed to explore such poten-

The association of lifespan with FOXO3A variants in several studies and the novel association we identified with a SNP in AKT1 strongly suggest that the same components of the insulin/ IGF1 signaling pathway that affect lifespan in C. elegans, Dro- sophila and mice are also involved in determining lifespan in humans. FOXO3A is one of the three mammalian homologues of the C. elegans transcription factor DAF-16, which is required for the lifespan effects of the insulin/IGF1 signaling pathway. AKT1 is one of the tyrosine kinases in the signaling cascade that links IGF1 binding to its receptor to transcriptional activation and downstream effects on cell metabolism and oxidative stress response; AKT1 phosphorylates FOXO3A and prevents it from entering the nucleus thus inhibiting transcriptional activation (Brunet et al., 1999; Zheng et al., 2000). Thus, the two genes most strongly associated with longevity in our results are also functionally related (Figure 1).

Our study has several potential limitations. First, although we adjusted for self-reported ethnicity in the SOF study, population stratification may have affected our results. In addition, we did not have self-reported ethnicity information in CHS. Second, subtle effects on longevity might have been missed because of our relatively small sample size (896 participants in SOF); we had adequate (80%) power to detect an odds ratio of (cid:2)1.7. As with other complex traits (Easton et al., 2007; Frayling, 2007; Harley et al., 2008), much larger sample sizes will be required to identify modest risk modifiers. Third, like most studies of long- evity in humans, our study may be influenced by birth cohort effects, as controls were generally born later (e.g. in the 1910s– 1920s) than the cases (in the 1890s–1900s). Finally, we only tested common SNPs and small insertion-deletions. We did not genotype copy number variants or large insertion-deletions and, with the exception of a few amino-acid coding variants, we only evaluated the effects of common variants (minor allele fre- quency > 0.05). The relative dearth of common SNPs associated with longevity in the genes we analyzed does not exclude the

How the variants we identified affect lifespan remains uncertain. All three longevity-associated SNPs that replicated across three independent cohorts are intronic SNPs of no known function, and they do not reside in motifs conserved through evolution. In the case of FOXO3A, the two longevity- associated SNPs are in LD with other variants across an area of approximately 67 kb (Fig. S1). As the LD is so strong in this

(A) (B)

(B) sfWindow Position Human Mar, 2006 chr14104303,701-104,310,700 (1,000 bp)Scale 500 bases|chia: 104310000] 104310500]RefSeq Genes          HOF IRINA,HeLa Cyto RNA.HeLa Nucl IRNA      Affymetrix Transcriptome Phase 3 Long RNA Signal‘SPERR Regulatory Potential (7= 2ifespan associated SNPsuh nllmu 0 Hy ll| ICpG Islands (Islands < 300 Bases are Light Green)X Reg Potential = a: mallr Vertebrate Multiz Alignment & PhastCons Conservation (28 Species)39603904 |C6 stands =Mammal Cons.