(forkhead box group O) transcription factors are targets of insulin-like signaling and are involved in a diverse set of physiological functions including DNA repair and resist- ance to oxidative stress [33,34]. Further, FOXO plays a role in lifespan extension in C. elegans and Drosophila [35]. Studies of this gene in humans are limited; two case-con- trol studies have not identified an association between FOXO1a and longevity [36,37]. However, the prospective population-based Leiden 85-plus Study found that FOXO1a was associated with increased mortality attribut- able to diabetes related deaths in participants aged 85 years and older [38]. The Leiden 85-plus Study also reported that genetic variation causing a reduction in insulin/IGF-1 signaling resulted in improved old age sur- vival among women [20]. However, that report examined other genes in the insulin/insulin-like signaling pathway and did not specifically examine FOXO1a. Finally, the untargeted approach to SNP selection also identified a SNP near FOXO3a associated with age at natural meno- pause. This gene has been implicated in oocyte death,

In our untargeted approach of ranking SNP by the strength of the p-value, 2 intronic FOXO1a SNPs were associated with age at death. One of these (rs4943794) also was associated with biologic age by in our a priori evaluation of select candidate genes.

depletion of functioning ovarian follicles, and infertility in mice [39,40] and thus represents a plausible candidate gene for menopause. Most positive common gene variant- disease association studies have failed replication [41] including reports on exceptional longevity. Haplotype- based fine mapping of the region on chromosome 4 linked to human longevity initially suggested the MTP gene, a gene important in lipoprotein synthesis, was asso- ciated with longevity [21]. However, this association failed replication in a French cohort of long-lived individ- uals and subsequent case-control studies of nonagenari- ans [22,42]. Beekman, et al [43] found neither linkage to chromosome 4 nor association with the MTP gene and longevity among nonagenarians in the Leiden Longevity Study. Meta-analyses implicated admixture of the control sample in the original report as an explanation for the pre- sumed false-positive association. Thus, our findings are hypothesis generating and their importance can not be determined without evidence of consistent replication in other populations.

Several important limitations merit comment. First, we acknowledge that there may be a survival bias as partici- pants in this sample had to survive to provide DNA (first systematic DNA collection began 1995) and hence are likely healthier than the full FHS sample. To ameliorate this issue, we adjusted for covariates using the full Fram- ingham sample, and used the residual traits for the subset of individuals genotyped using the 100K Affymetrix Gene- Chip to test for association with the SNPs using linear regression models. Residual traits from Cox and logistic models typically are not ideally distributed for linear regression models, but our adjustment method using the full sample precludes the testing of SNP associations with age at death and morbidity-free survival using Cox and logistic models. Second, the 100K Affymetrix GeneChip provides limited coverage of the genome; many of our a priori candidate genes did not have any SNP coverage on the chip. For example, several genes that have been stud- ied in model organisms or even in humans such as ACE, Lamin A, SIRT2 and SIRT3, had no SNPs within 60 kb of the gene on the 100K Affymetrix GeneChip. However, genotyping is near-complete for the NHLBI funded 550 K genome-wide scan on all FHS participants. This will ena- ble deeper exploration of our initial 100K SNP associa- tions in a larger sample with denser coverage of the genome. Third, in this analysis we did not examine epista- sis or gene-environment interactions which may modify the associations in this study. Importantly, this study is hypothesis generating. Our findings need to be replicated in other samples.

We examined pleiotropic effects by identifying SNP asso- ciations across two pairs of related traits. One SNP near PON1 emerged as associated with both age at death and morbidity-free survival. Surprisingly, there were relatively few SNPs associated with both traits; prior work had sug- gested that longevity per se and healthy aging may share common genetic pathways [11,12]. However, morbidity- free survival was measured at age 65 years, it is possible that as our participants age morbidity-free survival defined at age 75 or 85 years will share additional SNP associations with our longevity trait, age at death. A SNP near SOX5, a gene potentially related to musculoskeletal function was associated with both biologic age by OSS and walking speed.

Conclusion

In summary, the untargeted genome-wide approach to detect genetic associations with longevity and aging traits provides an opportunity to identify novel biologic path- ways related to lifespan control. GWAS also have the potential to direct investigators of human aging to the most promising candidate gene associations and biologic pathways reported to regulate lifespan in animal models. Enhancing our understanding of the mechanisms respon- sible for aging may in turn identify directions for health promotion and disease prevention efforts in middle-aged and older adults so that older persons can enjoy more time in good health. These data generate hypotheses regarding novel biologic pathways contributing to longev- ity and healthy aging and serve as a resource for replica- tion of findings from other population-based samples.