The significance threshold applied was selected by esti- mating the false-positive fraction for different significance thresholds P on simulated datasets. Data simulation was carried out by generating 100 independent datasets com- posed by 317K SNPs, with MAF distribution ranging from 0.05 to 0.50, and 963 control samples to which 410 ‘‘case’’ and 553 ‘‘control’’ labels were assigned randomly. We then tested for association between each marker and the case/control status assuming allelic, dominant, and recessive genetic models and selecting the most significant as the most infor- mative for each SNP. For each significance threshold (from p value 0.05 to p< 1(cid:2)10(cid:3)7) the mean fraction of false positive associations (a false positive association was defined as a SNP with p lower than the selected significance threshold P) was estimated over 100 simulated datasets for each P.

Determination of genotypes

Genotyping was carried out with the Infinium II Assay- HumanHap BeadChip 317K duo system (Illumina, San Diego, California) using standard protocols of the Illumina HumanHap 317 Duo workflow (Illumina, San Diego, CA). All genotypes were evaluated using a quantitative quality score called GenCall (GC) score. The GC score ranges from 0 to 1 and reflects the proximity within a cluster plot of the intensities of that genotype to the centroid of the nearest cluster.

Testing for association

Association between the set of SNPs passing the QC cri- teria and the longevity phenotype was tested by means of the 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 re- cessive genetic models was applied to estimate odd ratio (OR) and 95% confidence interval (CI) for each of the dif- ferent 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, respec- tively, 93% and 95%, as well as SNPs deviating from Hardy– Weinberg Equilibrium (HWE) ( p<1.72(cid:2)10(cid:3)7) or with a minor allele frequency (MAF) lower than 0.05 were re- moved. Principal component analysis (PCA) was applied on a reduced set of autosomal markers showing r2 estimates <0.2, using the default outliers removal threshold (sig- ma ¼6) 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 2(cid:2)106 cells/mL and infected with the appropriate lentivirus. Ex- pression 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 antic- oagulated 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 (cid:3)808C until the day of the assay. All procedures were performed in compliance with institutional guidelines for human research.26 Mononuclear cells were chosen on the basis of the previous knowledge that CAMKIV is expressed in these cells and on the assumption that a ge- notype/expression correlation is not tissue specific, thus re- flecting expression modification in all tissues that express CAMKIV.

tested with the low thresholds of significance that must be adopted.29 For this reason, even replicated associations such as FOXO3A and APOE can be missed if formal Bonferroni correction (that is, a correction of the significance of the p value on the basis of the number of tests performed) is ap- plied when analyzing these loci in a whole-genome context. In an attempt to generate a list of SNPs potentially associated with human longevity, we decided to screen SICS LLIs and controls. We genotyped these subjects with the Infinium II Assay-HumanHap 317K duo BeadChip (Illumina, San Die- go, CA), evaluating allelic, dominant, and recessive genetic association models.

Lentiviral infection. The lentiviral constructs were gen- erated and characterized by Kitsos et al.27 Briefly, CAMKIV- WT cDNA was cloned into a Lenti-IRES-GFP vector, and high-titer control and recombinant viruses were prepared by pseudotyping with vesicular stomatitis virus glycoprotein (VSV-G) using a quadruple transfection protocol in 293T cells.28 Approximately 2(cid:2)106 HT29cells 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 ev- idence 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).

Western blotting