The rs10491334 SNP was further tested for association in an independent replication set (SICSr) composed of 116 SICS LLIs (age range, 90–109 years; male/female ratio, 0.87; see Supplementary Table 1 for a detailed demographic and clinical characterization of SICS LLIs9) and 160 young con- trols (age range, 18–44 years; male/female ratio, 1.91) pass- ing the overall QC procedure. Power calculations using QUANTO software23 showed that we had intermediate power in both screening (70%) and replication (60%) steps with the sample sizes used and OR and frequency of rs10491334 (Supplementary Table 5). Association results on the SICSr cohort and the joint analysis of the screening and replication sets (516 LLIs vs. 713 controls) confirmed the association of rs10491334 (replication, OR¼ 0.54, 95% CI 0.32–0.90, p¼9(cid:2)10(cid:3)3; joint analysis, OR ¼0.55, 95% CI 0.43– 0.70, p¼1.68(cid:2)10(cid:3)6) (Table 2).

We reviewed the Disease Associated Catalog to determine whether any of our 67 selected SNPs had been previously associated with traits relevant to aging. We found that rs10491334 (calcium/calmodulin-dependent protein kinase IV [CAMKIV]) had been associated with high diastolic blood pressure in the Framingham Study (p ¼4.47(cid:2)10(cid:3)6).31 Asso- ciation tests on imputed SNPs confirmed rs10491334 with the strongest evidence of association within the CAMKIV locus (Supplementary Figure 5).

Association results were not influenced by gender- confounding effects (Supplementary Table 3). To be noted, we found a significant association for rs2075650 (TOMM40) (p< 0.05), a SNP inpartial LD with the APOe4 haplotype32that shows very low MAF (overall MAF of 0.05), in line with the observation that, among Europeans, relative APOE e4 fre- quencies exhibit a north-to-south cline.33 Furthermore, inde- pendent genetic analyses on the same population showed

To be noted, imputation analysis was explorative, subse- quent to the association analysis for rs10491334 and was done with the only purpose of understanding whether the signal detected at rs10491334 could have been explained by other nearby SNPs. LD analyses evidenced a haplotype block comprising rs10491334 and rs9326835 (Supplementary Fig. 6): multimarker association tests showed significant associ- ation for rs10491334[A]-rs9326835[G] haplotypes (OR¼ 0.56,

Table Association Results for rs 2. 10491334

No. (%) Alleles/genotypes Gene, SNP Study/phenotype a/A MAF Assoc Model LLIs Controls p valuea OR (95% CI) CAMKIV Dominant rs10491334 SICS/Long T/C 0.18 TT/TC vs. CC 104 (25) 211 (38) 2.88(cid:2)10(cid:3)5 0.55 (0.42–0.73) SICSr/Long T/C 0.19 TT/TC vs. CC 31 (27) 65 (41) 9(cid:2)10(cid:3)3 0.54 (0.32–0.90) SICS þSICSr/ Long T/C 0.18 TT/TC vs. CC 135 (26) 276 (39) 1.68(cid:2)10(cid:3)6 0.55 (0.43–0.70)

aReported p value for SICSr cohort was divided by two because only one direction of allele/genotype frequency difference was tested. Gene, Official gene symbol; SNP, Single-Nucleotide Polymorphism Database ID; Study/phenotype, SICS, Southern Italian Centenarian study, longevity; SICSr, Southern Italian Centenarian study replication set, longevity; SICSþSICSr, SICS screeningþreplication sets, longevity; a/A, minor allele/major allele based on whole samples; MAF, minor allele frequency; Assoc Model, associated genetic model (Dominant, dominant model [aa/aA vs AA], 1 df); No. (%) Alleles/Genotypes, number and percentage of reference alleles/genotypes (dominant, aA/AA) in long-living individuals (LLIs) and control individuals (controls); OR, odds ratio; 95% CI, 95% confidence interval. For example, subjects who carry the genotype TT/TC for rs10491334 (dominant model) have 0.55 times the odds for longevity compared to subjects who carry CC genotype.

lation.35 Therefore, the activation of AKT, FOXO3A, and SIRT1 reduces the activation of proapoptotic genes and favors cell cycle arrest and oxidative stress resistance.15 On the basis of this premise, we first investigated whether rs10491334 affected the protein expression of calcium/calmodulin-de- pendent protein kinase IV (CAMKIV). To this end, we used mononuclear cells of healthy controls carrying the polymor- phism in homozygosis (n¼ 10) and compared them to those from individuals carrying the wild-type gene (n¼10). We found that the protein level of CAMKIV is significantly lower in homozygous carriers (Fig. 2A). The reduction in CAMKIV protein expression driven by the polymorphism, together with

association between the APOE e4 allele (rs429358[C]-rs7412[C] haplotype) and longevity phenotype (p¼ 5.3(cid:2)10(cid:3)3).

CAMKIV and functional studies

Under growth factor stimulation, FOXO3A is phosphory- lated by AKT and binds to 14-3-3 proteins in the cytoplasm. Thus, it becomes unable to activate proapoptotic genes, such as the tumor necrosis factor (TNF) receptor superfamily member 6 (FAS).34 When oxidative stress induces transloca- tion of FOXO3A to the nucleus, its ability to activate proapoptotic genes is reduced by SIRT1-dependent deacety-

BCAMK4 WT CAMK4 rs10491334 CTR* MOCK CAMK4              ———__— ———_ MOUSE BRAIN pFOXO3A12 3 ABCSe eee FOXORAoo = WB CAMK4 TOT——— «meee = PSIRT!‘ » —) WB Actin SIRT1pAKTCAMK4 MONONUCLEAR CELLS —_© CAMK4 WT@® CAMK4 1810491334 comm OAMKA1.0= *aoO050.0Cc pFOXO3A pAKT50 aan© Mock . © Mock40 = camKa @ camk430a 2 6So OpSIRT1 CAMK4    i 20020 we © Mock © Mock=15 Mi camK4 150 CAMK42 =>a2B 10 2 1000.5 500.0 0FIG 2. Western blot analvsis of calcium /calmodulin-denendent protein kinase TV (CAMKTV). (A) Western blot analvs