Results from model organisms suggest the hypothesis that genes in the insulin/IGF1 pathway may be involved in the control of human lifespan, and variants that affect their activity may be associated with human longevity. Although a few associations of insulin/IGF1 pathway genes with longevity have been reported (Bonafe et al., 2003; van Heemst et al., 2005; Kuningas et al., 2007; Suh et al., 2008; Willcox et al., 2008), most of these findings have not been replicated and the studies did not analyze genetic variation in the insulin/IGF1 pathway systematically.

Table 2 Association of variants in insulin/IGF1 pathway genes with longevity

Gene SNPs P < 0.05 P < 0.01 P < AFX 7 0 0 0 AKT1 8 1 0 0 AKT2 5 0 0 0 AKT3 13 0 0 0 FOXO1A 7 1 0 0 FOXO3A 16 7 4 1 GHR 26 7 3 1 GHRH 1 0 0 0 GHRHR 13 1 0 0 IGF1 6 0 0 0 IGF1R 34 1 1 1 IGFBP1 11 0 0 0 IGFBP2 8 0 0 0 IGFBP3 5 1 0 0 IGFBP4 7 1 0 0 IGFBP5 11 0 0 0 IGFBP6 4 0 0 0 INS 2 0 0 0 INSR 32 0 0 0 INSRR 4 0 0 0 IRS1 6 0 0 0 IRS2 13 0 0 0 IRS4 1 0 0 0 PDPK1 3 0 0 0 PIK3CA 5 0 0 0 PIK3CB 3 0 0 0 PIK3R1 28 0 0 0 PIK3R2 2 0 0 0 PTEN 9 1 0 0 SHC1 1 0 0 0 All genes, observed 291 21 8 3 All genes, expected – 15 2.9 1.5

We have comprehensively evaluated common variation in genes in the insulin/IGF1 signaling pathway for association with human lifespan. We performed a nested case–control associa- tion study of longevity in a cohort of elderly Caucasian women from the Study of Osteoporotic Fractures (SOF). We then repli- cated significant associations in two independent Caucasian cohorts of men and women.

Results

All subjects were Caucasian. The SOF cohort was all women, the Cardiovascular Health Study (CHS) cohort was 47% female, and the Ashkenazi Jewish Centenarians (AJC) study was 66% female (Table 1). More than half (55.6%) of the longevity cases were still alive in SOF, about a third (33.7%) were still alive in CHS and 24% of cases were still alive in AJC.

Association of genotype with longevity in SOF

We evaluated 291 single nucleotide polymorphisms (SNPs) in 30 genes in the insulin/IGF1 signaling pathway (Figure 1, Table S1). Twenty-one (7%) SNPs in 9 (30%) genes (AKT1, FOXO1A, FOXO3A, GHR, GHRHR, IGF1R, IGFBP3, IGFBP4, and PTEN) were associated with longevity at a nominal P < 0.05, a modest excess over the expected (cid:2)15 (Table 2). Eight SNPs had a P < 0.01 (an excess of (cid:2)5 over the expected number), and three SNPs had P < 0.005 ((cid:2)1.5 more than expected). FOXO3A (7/16) and GHR (7/26) had the most SNPs associated with longevity.

NumberofSNPsineachgeneassociatedwithlongevityatanominally significantlevelisshown.Resultsshownareforanadditivemodelcorrected forself-reportedethnicity.TotalnumberofSNPsobservedatthreedifferent significancethresholdsiscomparedwiththenumberexpected.Genes withlongevity-associatedSNPshighlightedinbold.

haplotype-tagging SNP (data not shown). For GHR, we observed themostsignificantassociationforathree-SNPhaplotypecompris- ing rs9292854, rs13182117, and rs4866941 (P = 0.0005, com- pared with the best single-SNP association of P = 0.007 for rs4866941; analysis not corrected for race/ethnicity), suggesting thattheputativecausativealleleresidesonthishaplotype.

For all genes except GHR, haplotypes containing longevity-asso- ciated SNPs exhibited a similar effect size and significance level for association with longevity, suggesting that in each case, the puta- tivecausativevariantwasinlinkagedisequilibrium(LD)withasingle

we selected other variants in the surrounding area from dbSNP. We genotyped eight additional AKT1 SNPs in SOF, including five coding SNPs, (one rare, four unvalidated), two synonymous SNPs, and one intronic SNP located 22 bp from an intron–exon junction. Three of these eight SNPs were polymorphic in SOF and none was associated with longevity (Table S1). As no coding SNPs in AKT1 were associated with longevity, we searched for potential regulatory sites in the region (Fig. 2B). AKT1 rs3803304 lies in an intron, 70 bp away from a conserved exon– intron boundary and in an area of high-predicted regulatory potential (0.3 score on the evolutionary and sequence pattern extraction through reduced representations (ESPERR) prediction metric) (King et al., 2005). Data from the Affymetrix Transcrip- tome Phase 3 gene tiling arrays (Kapranov et al., 2007) show a long (> 200 bp) RNA signal at this intron/exon border encom- passing rs3803304 (Fig. 2B); a CpG island is also found here. It is possible that rs3803304 disrupts an RNA regulatory mechanism. Initial SNP discovery in FOXO3A was by sequencing—no other common exonic SNPs were found. The two SNPs most strongly

Replication of association of AKT1 and FOXO3A SNPs with longevity

As the case–control associations we observed in SOF were not sig- nificant after correction for multiple hypothesis testing, we repli- catedthenominallyassociatedSNPs intwo independentCaucasian cohorts, the CHS and AJC. We selected all 21 SNPs individually associated with longevity in SOF (P < 0.05) as well as 4 SNPs signifi- cantly associated with longevity (P < 0.001) in 2-SNP interaction analysesforreplicationgenotyping(Table3).Weobservedthemost consistent results across all three cohorts for 1 SNP in AKT1 (rs3803304), which was nominally associated with longevity in each of the three cohorts, with a consistent effect size (odds ratio, OR) of 0.77–0.79. In a meta-analysis of results from all three cohorts, this association was statistically significant (P = 0.0004; permutation-basedcorrectionformultipletestingP = 0.043).

Two SNPs in FOXO3A were nominally associated with longev- ity in SOF and in AJC, but not in CHS, although the magnitude and direction of the effect were consistent in CHS. The P-values in the meta-analysis were 0.0014 for rs1935949 and 0.0009 for rs4946935; these approached statistical significance in the meta-analysis after correction for multiple testing.