Summary 

The insulin/IGF1 signaling pathways affect lifespan in several model organisms, including worms, flies and mice. To investigate whether common genetic variation in this pathway influences lifespan in humans, we genotyped 291 common variants in 30 genes encoding proteins in the insulin/IGF1 signaling pathway in a cohort of elderly Caucasian women selected from the Study of Osteoporotic Fractures (SOF). The cohort included 293 long-lived cases (lifespan > 92 years (y), mean ± standard deviation (SD) = 95.3 ± 2.2y) and 603 average-lifespan controls (lifespan < 79y, mean = 75.7 ± 2.6y). Variants were selected for genotyping using a haplotype-tagging approach. We found a modest excess of variants nominally associated with longevity. Nominally significant variants were then observed in vertebrates, where growth and glucose metabolism are regulated by two parallel pathways with separate receptors for insulin and IGF1. In mice, mutations that result in growth hormone deficiency or mutationsThe most significant association for a three-SNP haplotype comprising rs9292854, rs13182117, and rs4866941 (P = 0.0005, compared with the best single-SNP association of P = 0.007 for rs4866941; analysis not corrected for race/ethnicity), suggests that the putative causative allele resides on this haplotype. For all genes except GHR, haplotypes containing longevity-associated SNPs exhibited a similar effect size and significance level for association with longevity, suggesting that in each case, the putative causative variant was in linkage disequilibrium (LD) with a single SNP. 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 Transcriptome Phase 3 gene tiling arrays (Kapranov et al., 2007) show a long (> 200 bp) RNA signal at this intron/exon border encompassing 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 associated with longevity, FOXO3A rs1935949 and rs4946935, lie in intron 3, 1.7 kb from the intron-exon boundary of the 3UTR, but not in an area of evolutionary conservation (Fig. 2D). We observed a borderline effect of sex on the FOXO3A rs1935949 SNP association with longevity, suggesting a stronger effect of female sex (OR = 0.72, 95% CI = 0.52-0.99, P = 0.049), as also seen when comparing the odds ratio for association with longevity between the mixed-sex (OR = 1.24, 95% CI = 1.09-1.41) and female-only subsets (OR = 1.35, 95% CI = 1.15-1.57). When restricted to female subjects, the association of FOXO3A rs1935949 and rs4946935 with longevity in the meta-analysis achieved statistical significance (adjusted P-values were 0.0093 and 0.019 respectively).

Interaction analyses

We included four SNPs in the replication genotyping that were not individually associated with longevity in SOF but had a significant association with longevity in interaction analyses with another SNP. Two IGFBP5 SNPs (rs3770472 and rs11575134) had significant interactions with GHR SNP rs2940935 in SOF (both P < 0.00004); these interactions appeared to replicate in the AJC study (P = 0.012 for rs3770472 and 0.026 for rs11575134), but not in the CHS cohort. SNP rs1009728 was included in the replication genotyping because of a significant interaction with a SNP in PIK3R1, but this latter SNP could not be genotyped in the replication cohorts.

Replication of association of AKT1 and FOXO3A SNPs with longevity

As the case-control associations we observed in SOF were not significant after correction for multiple hypothesis testing, we replicated the nominally associated SNPs in two independent Caucasian cohorts, the CHS and AJC. We selected all 21 SNPs individually associated with longevity in SOF (P < 0.05) as well as 4 SNPs significantly associated with longevity (P < 0.001) in 2-SNP interaction analyses for replication genotyping (Table 3). We observed the most 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-analysis42.022.159.080.153.053.198.001.187.082.128.030.134.063.178.090.14PBFGInoitcaretnI8279001sr 490.020.197.009.073.021.137.019.086.082.148.040.1210.039.095.057.04PBFGIytivegnoL6644127sr 63.042.139.070.1340.036.100.182.104.051.107.009.017.073.108.050.15PBFGInoitcaretnI43157511sr 06.002.

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