Gene and variant selection

Variants were selected for genotyping in 30 genes encoding the key proteins (Figure 1) in the human insulin/IGF1 signaling path- way, including ligands (IGF1, insulin, GH1, GHRH), binding pro- teins (IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP6), receptors (IGF1R, INSR, INSRR, GHR, GHRHR), signal transduc- tion proteins (AKT1, AKT2, AKT3, IRS1, IRS2, IRS4, PDPK1, PIK3CA, PIK3CB, PIK3R1, PIK3R2, PTEN, SHC1), and transcrip- tion factors (AFX, FOXO1A, FOXO3A). GH1 was included ini- tially, but all three SNPs available in dbSNP failed genotyping attempts. Initially, variants in FOXO1A and FOXO3A were identi- fied by sequencing exons, intron–exon boundaries, and the pro- moter in 80 Caucasians and selecting haplotype-tagging variants. Subsequently, we utilized the International HapMap Consortium data from CEU Caucasians (The International HapMap Consortium 2005; Frazer et al., 2007) (http:// www.hapmap.org) to select haplotype-tagging SNPs. Briefly, tag SNPs were picked for the gene ± approximately 10 kb genomic flanking sequence using the Tagger algorithm (de Bakker et al., 2005) implemented in the Haploview pro- gram (Barrett et al., 2005) (http://www.broadinstitute.org/mpg/ haploview). The entire genomic sequence was tagged for all genes except IGF1R and INSR, where most of the large intron 1 was excluded. We set the r2 cutoff at 0.8 and minor allele fre- quency cutoff at 0.05 (except for amino-acid coding variants, which were all included). Pair-wise tag selection was used for all genes except IGFBP2, IGFBP5, IRS2, and PIK3R1, where two- marker haplotypes were used to reduce the number of tag SNPs. Amino-acid coding variants were prioritized and previously pub- lished candidate variants were included (Bonafe et al., 2003). Eight additional SNPs in AKT1 were selected for follow-up. We selected 339 SNPs overall; 293 SNPs were successfully geno- typed.

Single nucleotide polymorphisms genotype distributions in SOF controls were tested for consistency with Hardy–Weinberg equilibrium using a c2 test (Bonferroni-corrected significance threshold P = 1.7E)04). 2/293 SNPs, IRS4 rs1801162 and IGFBP2 rs9341156, were not in Hardy–Weinberg equilibrium and were removed from further analysis.

Statistical analyses

of SNP genotype with longevity

For all 291 SNPs analyzed, minor allele homozygotes were coded 2, heterozygotes were coded 1, and major allele homozygotes were coded 0. Logistic regression was used to evaluate the asso- ciation between genotype and longevity case/control status. We assumed an additive model as our baseline model; significant associations were also tested with a genotypic model. In SOF, the logistic regression model was adjusted for self-reported ethnicity. In CHS and AJC, models were adjusted for sex; we also analyzed data separately for women in these two cohorts. Asso- ciation of significant genotypes with the three main categories of cause of death (cardiovascular disease, cancer, or other) was evaluated in the two cohorts (SOF and CHS) that had this infor- mation. OR and 95%CI are reported. We also performed haplo- type case–control association analyses using two- and three-SNP sliding window and haplotype analysis across entire LD-blocks using the Haploview program (Barrett et al., 2005).

To analyze the genomic regions surrounding FOXO3A and AKT1, we used the UCSC Genome Browser (http://www.geno- me.ucsc.edu) including data from several analysis tracks (ESPRR regulatory potential (King et al., 2005), Phastcons conservation, CpG islands, Affymetrix Transcriptome Phase 3 (Kapranov et al., 2007)) on the March 2006 assembly of the human genome.

Replication and meta-analysis

The logistic regression results from all three cohorts (or from two cohorts only in some cases) were combined using inverse variance weighting. To evaluate statistical significance in the replication, we corrected for all of the SNPs we replicated and used only the data from the two replication cohorts (AJC + CHS) to assess significance. We determined statistical significance by permutation. Case/control status was permuted randomly for all participants for the 25 SNPs genotyped in the replication stage. In each permutation, we recorded the maxi- mum absolute value of the Z scores. We performed 2000 permutations. We then compared the Z scores from the

Genotyping

Single nucleotide polymorphisms were genotyped in the SOF and CHS cohorts using two genotyping platforms: template directed primer extension with fluorescence polarization detection (FP- TDI, AcycloPrime II detection kit, Perkin Elmer, Boston, MA, USA) (Hsu & Kwok, 2003) and SNPstream 48plex (Beckman Coulter, Fullerton, CA, USA). Oligonucleotide primers were designed

observed data with the distribution of the maximum Z scores from the permutations. The percentile of the observed absolute z score among the permuted z scores was calculated as the adjusted P-value for determining statistical significance cor- rected for multiple testing. Statistical significance in the replica- tion was defined as an adjusted P < 0.05 in the AJC + CHS replication cohorts.

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