Testing for association: Association between the set of SNPs passing QC criteria and longevity phenotype was tested by means of one degree of freedom (df) Pearson chi-squared allelic association test (a [minor allele] vs. A [major allele]) and assuming a dominant (aa/aA vs. AA, 1 df) and recessive genetic effect (aa vs. aA/AA, 1 df). Logistic regression assuming additive, dominant, and recessive genetic models was applied to estimate odd ratio (OR) and 95% confidence interval (CI) for each of the different genetic models, to test for association in the replication set, and to evaluate potentially confounding gender-specific effects. Adjustment for potential confounding effects due to residual genetic stratification was done with the genomic control approach.24

Quality control analyses: Samples and SNPs with a Call Rate lower than 93% and 95%, respectively, as well as SNPs deviating from Hardy Weinberg Equilibrium (HWE) (p<1.72x10^-7) or with a minor allele frequency (MAF) lower than 0.05 were removed. Principal component analysis (PCA) was applied on a reduced set of autosomal markers showing r2 estimates <0.2, using the default outliers removal threshold (sigma 6) as implemented in Eigenstrat software.20 All statistical procedures were performed using PLINK,25 except when specified.

In vitro evaluation of CAMKIV: Imputation of unobserved genotypes: Cell cultures. Human colon adenocarcinoma grade II HT29 cells were maintained in Dulbecco modified Eagle medium (DMEM) with 10% fetal bovine serum. All media were supplemented with 100 units/mL penicillin and 100 mg/mL streptomycin. The cells were seeded at 2x10^6 cells/mL and infected with the appropriate lentivirus. Expression of CAMKIV-WT was confirmed by western blot using a specific antibody against CAMKIV. SNPs that were not represented on the Illumina Infinium II Assay-HumanHap BeadChip 317K were imputed by the Impute v2.1.2 software,21 using the 1,000 Genomes files from CEU (Centre d'Etude du Polymorphisme Humain [CEPH] [Utah residents with ancestry from northern and western Europe]) genotypes data (release March 2010) as reference panel.

Linkage disequilibrium analyses: Mononuclear cells. Blood was collected and anticoagulated with EDTA. Mononuclear cells were isolated by Ficoll gradient using HISTOPAQUE-1077 (Sigma, USA). Linkage disequilibrium (LD) analyses were performed using Haploview v4.2 software.22 Haplotype blocks were frozen and stored at -80°C until the day of the assay. All procedures were performed in compliance with institutional guidelines for human research.26 Mononuclear cells were chosen based on previous knowledge that CAMKIV is expressed in these cells and on the assumption that a genotype/expression correlation is not tissue-specific, thus reflecting expression modification in allBy pseudotyping with vesicular stomatitis virus glycoprotein (VSV-G) using a quadruple transfection protocol in 293T cells, approximately 2x106 HT29 cells were infected with the appropriate lentivirus: CAMKIV-WT (CAMKIV), or empty lentivirus (mock) as a control. The initial screening dataset was composed of 466 LLIs, 624 controls, and 318,237 SNPs. After a preliminary phase of QC, as described in the Methods section, we looked for evidence of genetic population stratification on a subset of 454 LLIs and 591 young controls by applying PCA20. A graphical inspection of the first two principal components revealed genetic homogeneity between LLIs and controls (Fig. 1). 

After outliers were removed (n=82), 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 phospho-AKT (Ser473) were purchased from Cell Signaling technology. Monoclonal CAMKIV antibody was purchased from BD Biosciences (San Diego, CA). One-way analysis of variance (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 ± standard error of the mean (SEM) of at least three independent experiments.

We then tested for association between each of the identified SNPs and longevity phenotype. Inspection of the quantile-quantile plots (Supplementary Fig. 1) and the estimated genomic inflation factor (λ)24 for the different genetic models revealed a moderate residual effect of population stratification on the association results (allelic λ, 1.09; dominant λ, 1.07; recessive λ, 1.00). Therefore, we decided to remove this potential confounding factor by genomic control adjustment24.

Simulation results (see Methods) showed that the rate of false-positive findings was a function of the significance inclusion cutoff, suggesting that the choice of p < 1x10^-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 does not rely on choosing the right gene beforehand, they do require a large sample size to support with adequate power the hundreds of thousands of SNPs.