Supportive evidence is beginning to accumulate for a role of insulin-signaling in human aging and longevity, but the genes that might mediate these effects are not known. Prior studies have found over or under representation of single nucleotide polymorphisms (SNPs) from the insulin-IGF-1signaling pathway in long-lived hu- mans of Italian (36), Japanese (37, 42), Dutch (60), and Ashkenazi Jewish (38) ethnicity, with links to several aging phenotypes. While some of these findings have been limited by small effect sizes and marginal statistical significance, the study by Suh et al. (38) also demonstrated that functionally significant mutations in the IGF-1

Subjects and Methods

Study Population. This nested-case control study was conducted as part of the HawaiiLifespanStudy,anembeddedcohortstudyofhealthyagingdrawnfrom theoriginalpopulationoftheHonoluluHeartProgram(HHP)andHonoluluAsia Aging Study (HAAS). The HHP is a population-based, prospective study of car- diovasculardiseaseamong8006JapaneseAmericanmenthatbeganin1965.The HHP participants were recruited from 9877 men with valid contact information who were born from 1900–1919 and living on the island of Oahu (61).

StudyparticipantshadbothparentsfromJapan,usuallythewestandsouthern parts of Japan (61, 62). Although most participants were born in Hawaii (88%), thereisatheoreticalpossibilityofconfoundingofcasecontrolstatuswithallele frequencies due to geographic origin. Therefore, for certain analyses, cases and controlswerestratifiedbyparentalprefectureoforiginusingconditionallogistic regressionmodels.Analysesshowednoevidenceforpopulationstratificationin thedataset(datanotshown).TheHHPcohorthasbeendescribedelsewhere(62). Briefly, surveillance of hospital records, obituaries, and national databases has resulted in extensive morbidity and mortality data (adjudicated by an expert committee) and near complete follow-up (61–63).

To date, there has been little study of FOXO genes and pheno- types of aging in humans. Two recent studies suggest that FOXO genes deserve further scrutiny. First, a longitudinal study of elderly Dutch men and women found that a FOXO1A haplotype predicted 4-year survival and that a FOXO3A haplotype predicted stroke risk (39). Second, the Framingham Study, in a genome-wide association analysis, found that a FOXO3A SNP was strongly associated with aol)(  age at natural menopause in women (P 0.00003). However, the Dutch findings were not statistically significant when accounting for multiple comparisons and both studies need replication. The present study is supportive and extends the associations of FOXO3A to human longevity and insulin sensitivity.

All participants for the current study were drawn from records of study participants updated to August, 2007. Archived phenotypic data and blood samples from Examination 4 of the HHP (1991–1993), which coincided with the commencement of the Honolulu Asia Aging Study (HAAS), was used as the baselineexaminationforthisnestedcase-controlstudy.TheHAASwasbegunas anexpansionoftheHHPforthestudyofneurodegenerativediseases,cognitive function, and other aging phenotypes in elderly persons (64). Participants in- cluded 3741 men aged 71 to 93 at Examination 4 (mean age 77.9 a. 4.7 years), approximately half the number of the original HHP (64).

One of the major advantages of the current study is that it used a nested case-control design. This study design selects cases and controls from an ongoing cohort study with longitudinally collected data. Therefore, several phenotypes of interest (e.g., disease prev- alence, health status, function) were obtained by direct clinical examination when the participants were younger, making the data less subject to recall bias.

For the purposes of the current nested case-control study, ‘‘cases’’ (longevity phenotype)weredefinedasallHHPparticipantswhohadsurvivedtoatleastthe upper1%ofthe1910U.S.birthcohortspecificsurvival(minimum95yearsofage) fromthetimeofrecruitment(65,66).Atotalof213individualswhohadsurvived toatleast95yearsofage,asofAugust2007,werestudied.Oftheseindividuals, 176 had died (mean death age 97.5; SD 2.1; range of 95–106 years) and 37 individuals were still alive (mean age 98.7, SD 2.1; range 97–106 years).

Indeed, studies of exceptional survivors, such as centenarians, that have found evidence for phenotypes suggestive of slower aging (46–48) could potentially suffer from significant recall bias. That is, older participants may not recall precisely their past medical history and their past functional status. However, in the current study, major diseases were adjudicated by a morbidity and mortality committee and performance-based measures of physical and cog- nitive function were used to supplement self-reports, and evidence was found for such a healthy aging phenotype. This lends prospec- tive support to previous retrospective work.

Thecontrolsconsistedof402individualsfromtheHHP/HAAScohortwhodied near the mean death age for the 1910 U.S. birth cohort specific survival for middle-agedmen((cid:8)77yearsofage).Toachieveacase:controlratioof(cid:8)1:2,we sampledtheHHP/HAASstudypopulationforcontrolswhodieduptotheageof 81years.Themeanageatdeathforourcontrolpopulationwas78.5years(SD1.8, range 73–81 years). This is slightly higher that of the U.S. male population, but consistent with the high average life expectancy of Japanese-American men in Hawaii,whichwas3.5yearslongerthanwhitemalesatlastreport(67).Allwere ethnic Japanese (61, 62).

Procedures were performed according to institutional guidelines and ap- proved by the Institutional Review Board of Kuakini Medical Center. Written informed consent was obtained from all study participants or from family rep- resentatives if participants could not provide consent.