Western blotting

After outliers were removed (n ¼82), the final GWAS dataset was composed of 963 samples, of which 410 were cases (age range, 90–109 years; male/female ratio, 1.47; see Supplementary Table 1 for a detailed demographic and clinical characterization of SICS LLIs9) and 553 were controls (age range, 18–48 years; male/female ratio, 1.56). A total of 298,715 SNPs passed the overall QC criteria.

For western blot analysis, mouse and rabbit monoclonal antibodies to SIRT1 and anti-phospho-SIRT1 were purchased from Abcam (Cambridge, UK). Polyclonal antibodies to FOXO3A, phospho- FOXO3A (Ser253), AKT, and pospho- AKT (Ser473) were purchased from Cell Signaling technol- ogy. Monoclonal CAMKIV antibody was purchased from BD Biosciences (San Diego, CA). One-way analysis of vari- ance (ANOVA) was performed to compare different groups using Bonferroni post hoc analysis. A significance level of p <0.05 was assumed for all statistical evaluations. Statistics were computed with GraphPad Prism Software (San Diego, California). Each value in the figures represents mean (cid:4)standard error of the mean (SEM) of at least three independent experiments.

We then tested for association between each of the iden- tified SNPs and longevity phenotype. Inspection of the quantile–quantile plots (Supplementary Fig. 1) and the esti- mated genomic inflation factor (l)24 for the different genetic models revealed a moderate residual effect of population stratification on the association results (allelic l, 1.09; domi- nant l, 1.07; recessive l, 1.00). Therefore, we decided to re- move this potential confounding factor by genomic control adjustment.24

Results

Simulations results (see Methods) showed that the rate of false-positive findings was a function of the significance in- clusion cutoff, suggesting that the choice of p <1(cid:2)10(cid:3)4 for at least one of the evaluated genetic models as the significance would guarantee a false-positive rate of approximately 2 in 10,000 independent tests (Supplementary Fig. 2).

Association study

Although GWAS do not rely on choosing the right gene beforehand, they do require a large sample size to support with adequate power the hundreds of thousand of SNPs

LLIs + ControlsControlsLLIssto Orosto Orosto Orozzso’;0 §=6©00';0 )=s0'0-  soo) §=6©000 = S0'0-Od soo 000 s00-Odre)So°Sore)=So-0.05 0.000.05 0.10 0.15PC1-0.05 0.000.05 0.10 0.15PC1-0.05 0.00PC1

FIG. 1. Population structure of long-living individuals (LLIs) (þ) and control subjects (D). Each scatter plot shows the first two principal components that were estimated using genotype data for Southern Italian Centenarian Study (SICS) LLIs and control subjects using the EigenSoft program. The two populations are ethnically identical, as shown in the right scatter plot.

Table Associated with Longevity in 1. SNPs SICS LLIs