Fig. 2 Genomic context and linkage disequilibrium for (A, B) AKT1 and (C, D) FOXO3A. (A) Linkage disequilibrium plot for AKT1 of r2 in HapMap Caucasians. HapMap SNPs at MAF > 5% are shown. SNPs genotyped in SOF are marked by black triangles. The longevity associated SNP rs3803304 is marked by a white triangle. (B) Close-up of 1kb area around rs3803304. UCSC genome browser tracks show mRNA isoforms, expressed RNAs, CpG islands, evolutionary and sequence pattern extraction through reduced representations regulatory potential (ESPRR) and conservation across 28 mammalian species. (C) Linkage disequilibrium plot for FOXO3A of r2 in HapMap Caucasians, showing all SNPs genotyped in SOF. The longevity- associated SNPs rs1935949 and rs4946935 are marked by numbers 141 and 142. A more detailed view showing all HapMap SNPs in the region is shown in Supplementary Figure 1. (D) Close-up of 8 kb area around rs1935949 and rs4946935. UCSC genome browser tracks show mRNA isoforms, expressed RNAs, regulatory potential (ESPRR)and conservation across mammalian species.

(C) (D) C) orsseed % Leadon ioahick toaldek tomdom MontaR SomlOR t Taine Teale aloo. Semisrary yeaa 7 fo sigeun gues + qatagy Mae 8 a8 phageEntrez genes =—a——» FORDOA: Ferknead box 03WOOoo ?—FOIA: Fortead box OR        tL L. I mae ]Lu tt 5 ZA TI2 5 2 8= s i 2= = &2 : = Boe 3‘)2   2 D) ‘Window Position Human Mar, 2006 chr6:109,104,801-109,112,800 (8,000 bp)Scale 2kb)che: | 103110000)RefSeq GenesFOXO3Toone est TELA L065 651 eeimhiaeiheasiieleeneieinFOXO3A Mespan-associated SNPs:rsisass49] rsagaea35|A ee \ :TE in wnt sESPERR Regulatory Potential (7 Species.ll a ll llVertebrate Multiz ‘hh id Conservation (28 Species)Nenmascors WOMLl dh etd tee wih Ld \, 4HOF IRNAHeLa Cyto RNAHeLa Nucl RNA 7X Reg Potential 

existence of other genetic effects in the pathway. To obtain a complete picture of the association of genetic variation in the insulin/IGF1 pathway with longevity phenotypes, discovery of rare variants by re-sequencing of the same set of candidate genes will be necessary.

because both groups were born before there were substantial changes in early-life mortality rates due to advances in medical care.

In summary, we screened the insulin/IGF1 signaling pathway for association with human longevity and identified common variants in two genes, AKT1 and FOXO3A, which are consis- tently associated with longer lifespan in three independent Caucasian cohorts. Our results extend the remarkably consistent effect of this pathway on lifespan in model organisms to human populations. Although the effects we observed are subtle, they implicate the insulin/IGF1 signaling pathway in human aging, confirming that studies in model organisms can help us to gain insights into the biology of human aging.

Despite these limitations, replication among three indepen- dent Caucasian samples, including a founder population (AJC), strengthens our findings. It is unlikely that population stratifica- tion could lead to false-positive results that would be consistent across a European American/Caucasian American population and the Ashkenazi population, as the patterns of stratification would be different. Any birth cohort effects should be relatively the modest, as the differences in birth-year were small, and

Cardiovascular Health Study (CHS)

Experimental procedures

The original CHS cohort includes 5201 Caucasian and African- American participants recruited from 1989 to 1990 in four com- munities in the United States (Fried et al., 1991; Tell et al., 1993) and 684 additional African-American participants recruited from 1992 to 1993. All participants were 65 years of age or older. At enrollment, participants were not institutionalized, wheelchair- bound at home, or receiving treatment for cancer. Informed consent was obtained from all participants. Deaths were ascer- tained through surveillance and at semiannual contacts. For all deaths, the CHS collected information from medical records, physician interviews, autopsy reports, coroner reports, and next of kin interviews. Data were reviewed by the CHS Events Com- mittee and cause of death classified according to prespecified criteria (Ives et al., 1995). From the 4383 Caucasian CHS partici- pants, we defined a case–control replication sample of 1076 for the longevity analysis using the same age cutoffs as in the SOF study. This subset included 279 long-lived cases (lifespan ‡ 92 year, mean = 94.5 ± 2.1 years) and 797 average-lifespan controls (lifespan £ 79 year, mean = 76.1 ± 3.1 year).

Samples

Data from three studies were analyzed. Two are large cohort studies of older adults: SOF and CHS. The third study is a case– control study (AJC).

Study of Osteoporotic Fractures (SOF)