In conclusion, all published nonsynonymous (coding and resulting in amino acid change) SNPs identified in the FOXO3 gene, to date, are either invalid or too rare to be of consequence to human longevity. The novel variant that we identified through detailed sequencing appears to be too low in frequency (0.082) to be responsible for our previously reported association of FOXO3 variants with human longevity (9), although it is conceivable that this novel SNP is associated with a disease phenotype at a younger age or is important in other populations. This suggests that the functional SNP is most likely a noncoding SNP from intron 2, where all previously reported associations have occurred. Future research should include complete sequencing of intron 2 and annotation of regulatory elements, in order to identify candidates for functional study of the FOXO3 gene and human aging.

Funding: We appreciate the support of contract N01-AG-4-2149, grants 5 U01 AG019349-05 and 2R01 AG027060-05A2 (Hawaii Lifespan Study) from the National Institute on Aging and contract N01-HC-05102 from the National Heart, Lung, and Blood Institute.

Acknowledgments: Author contributions: T.A.D., B.J.W., Q.H., J.S.G., K.H.M., B.R., and J.D.C. designed research; T.A.D., B.J.W., Q.H., R.C., J.S.G., K.H.M., B.R., and J.D.C. performed research; T.A.D. contributed new reagents and analytic tools; Q.H. and J.S.G. analyzed data; and T.A.D., B.J.W., Q.H., J.S.G., K.H.M., B.R., D.C.W., and J.D.C. wrote the article.

In the future, when more evidence accumulates as to a functional SNP(s), rigorous assessment with model organisms and human studies will be needed. Work with model organisms has already provided some clues that might be helpful for mechanistic studies of the effects of FOXO3 variation in humans. For example, oxidative stress, still a leading theory of how we age (35,36), is one potential mechanism. Indeed, the PI3K/Akt signaling cascade, a downstream target of insulin signaling, is an important inhibitor of FOXO function. Important antioxidant enzyme expression, including manganese superoxide dismutase (MnSOD) and catalase, depends on PI3K/Akt/FOXO3 activity (37,38), and oxidative stress appears linked to human longevity (39). Yet, work to date shows that mice deficient in the FOXO targetStudies: Contributors to Our Understanding of the Aging Process and Current Gerontology Geriatric Research. Barzilai N, Atzmon G, Schechter C, et al. Unique lipoprotein and genotype associated with exceptional longevity. JAMA. Density lipoprotein cholesterol, cholesteryl ester transfer protein gene variants, and healthy aging in very old Japanese-American men. J Gerontol A Biol Sci Med Sci. ( ). Estivill X, Cheung J, Pujana MA, Nakabayashi K, Scherer SW, Tsui LC. Chromosomal regions containing high-density and ambiguously mapped putative single nucleotide polymorphisms (SNPs) correlate with segmental duplications in the human genome. Hum Mol Genet. ( ). Musumeci L, Arthur JW, Cheung FS, Hoque A, Lippman S, Reichardt JK. Single nucleotide differences (SNDs) in the DNA Polymorphism database may lead to errors in genotyping and haplotyping studies. Hum Mutat. ( ). Manolio TA, Collins FS, Cox NJ, et al. Finding the missing heritability of complex diseases. Nature. ( ). ENCODE Project Consortium. The ENCODE (Encyclopedia of DNA Elements) Project. Science. ( ). Banasik K, Ribel-Madsen R, Gjesing AP, et al. The FOXO A rs G-allele associates with improved peripheral and hepatic insulin sensitivity and increased skeletal muscle-FOXO A NA expression in twins. J Clin Endocrinol Metab. ( ):E Beckman KB, Ames BN. The free radical theory of aging matures. Physiol Rev. ( ). Salmon AB, Richardson A, Perez VI. Update on the oxidative stress theory of aging: does oxidative stress play a role in aging or healthy aging? Free Radic Biol Med. ( ). Brunet A, Bonni A, Zigmond MJ, et al. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell. ( ). Kops GJ, Dansen TB, Polderman PE, et al. Forkhead transcription factor FOXO a protects quiescent cells from oxidative stress. Nature. ( ). Suzuki M, Willcox DC, Rosenbaum MW, Willcox BJ. Oxidative stress and longevity in Okinawa: an investigation of blood lipid peroxidation and tocopherol in Okinawan centenarians. Curr Gerontol Geriatr Res. ( ). Zhang Y, Ikeno Y, Qi W, et al. Mice deficient in both Mn superoxide dismutase and glutathione peroxidase- have increased oxidative damage and a greater incidence of pathology but no reduction in longevity. J Gerontol A Biol Sci Med Sci. ( ). Jang YC, Perez VI, Song W. et al. Overexpression of Mn superoxide dismutase does not increase lifespan in mice. J Gerontol A Biol Sci Med Sci. ( ). Ferber EC, Peck B, Delpuech O, Bell GP, East P, Schulze A. FOXO a regulates reactive oxygen metabolism by inhibiting mitochondrial gene expression. Cell Death Differ [published online ahead of print December ]. doi:/cdd. Yang JY, Hung MC. A new fork for clinical application: targeting forkhead transcription factors in cancer. Clin Cancer Res. ( ). Greer E, Brunet A. FOXO transcription factors at the interface between longevity and tumor suppression. Oncogene. ( ). Lebel M, Picard F, Ferland G, Gaudreau P. Drugs, nutrients, and phytoactive principles improving the health span of rodent models of human age-related diseases. J Gerontol A Biol Sci Med Sci. ( ).