RESULTS

Genome-wide association analysis

In order to identify novel loci involved in lifespan regulation, we conducted a meta-analysis on GWAS data of 7729 long-lived cases (≥ 85 years) and 16 121 younger controls (,65 years) from 14 studies originating from 7 European countries (Supple- mentary Material, Table S1). For each study, cases and controls originated from the same country. Given the higher heritability of longevity at older ages (11,20), we performed a subset ana- lysis in which we compared cases aged ≥ 90 years (n ¼ 5406) with 15 112 controls (,65 years) from the corresponding control cohorts. Replication was performed in 13 060 cases aged ≥ ≥ 85 years (of which 7330 were 90 years) and 61 156 controls from 6 additional studies, of which 3 originated from European countries not represented in the discovery phase meta-analysis (Supplementary Material, Table S1). Analysis of each study was performed using a logistic regression-based method, and results were adjusted for study-specific genomic inflation factors (l) (Supplementary Material, Table S2). Meta-analysis ≥ ≥ was performed on 2 480 356 ( 85 years) and 2 470 825 ( 90 years) imputed SNPs using a fixed-effect approach, and results

Prospective analysis

To determine the association of rs4420638 (TOMM40/APOE/ APOC1 locus) and rs2149954 (chromosome 5q33.3 locus) with longitudinal survival, we performed a prospective meta-analysis of the 2 SNPs in 34 103 individuals aged 30– 105 years from 11 different cohorts, of which 8582 had died after a mean follow-up time ranging from 2.2 to 17.4 years

                                           ( \ 7729 cases 2 85 years of age [14 studies] 5406 cases > 90 years of age [12 studies]16121 cons <65 years 15112 = <65 yearsAutosomal HapMap 122 SNPsMAF > 1% (or = 5% for small studies (< 200 cases))Discovery R,2>40Fixed effect approach9 loci with > 1 SNP with P< 1 x 10% 12 loci with > 1 SNP with P< 1 x 1054 ; ;- [ 13060 cases > 85 years of age [6 studies] [ 7330 cases > 90 years of age [6 studies]61156 controls < 65 years 61156 controls < 65 yearsReplication| 13 loci successfully genotyped with Sequenom |\ J 2 loci only replicated in cohorts with GWAS data( >) (" cases > 85 years of age [20 studies] | 12736 cases > 90 years of age [18 studies]77277 controls < 65 years 76268 controls < 65 yearsJoint analysis LOMMAUARORAROG! TOMM40/APOE/APOC1ORE 082.2 52.53010" OR = 0.72, P= 3.40 x 10°Chromosome 5q33.3OR = 1.10, P= 1.74 x 10%Prospective analysis in 34103 individuals (8582 deaths) [11 cohorts]30 - 105 yearsFollow-up TOMM40/APOE/APOCIHR = 1.07, P= 0.019Chromosome 5q33.3HR = 0.95. P= 0.003

Figure1.Flowchartofexperimentalwork.Theanalysisinthecasesaged ≥90yearsisasubsetanalysisoftheanalysisinthecasesaged studiesusedforthediscoveryphaseanalysisofcasesaged cases aged ≥90 years.

(Supplementary Material, Table S5). Carriers of the minor allele of rs4420638 (G) showed significantly higher all-cause mortal- ity (hazard ratio (HR) ¼ 1.07, P ¼ 0.019), whereas carriers of the minor allele of rs2149954 (T) demonstrated significantly lower all-cause mortality (HR ¼ 0.95, P ¼ 0.003; Supplemen- tary Material, Table S6).

P ¼ 2.27 × 1027). In addition, we observed an effect of this SNP on non-CVD mortality (HR ¼ 0.86, P ¼ 0.002) (Supple- mentary Material, Table S7). We also examined the Coronary ARtery DIsease Genome-Wide Replication And Meta-Analysis (CARDIoGRAM) GWAS (23), which showed a significant association of rs2149954 with a decreased risk for coronary artery disease (CAD) (OR ¼ 0.96, P ¼ 0.011) (Supplementary Material, Table S8). In addition, two SNPs on chromosome 5q33.3 in high LD with rs2149954, rs9313772 (r2 ¼ 0.928) and rs11953630 (r2 ¼ 0.854) have previously been reported to associate with blood pressure and hypertension (24,25). As expected, examining rs2149954 in the International Consortium for Blood Pressure GWAS (24) showed a significant association of the minor allele with lower diastolic (P ¼ 3.46 × 1025) and systolic (P ¼ 6.55 × 1026) blood pressure (Supplementary

Association with cardiovascular disease and blood pressure

To gain insight into the mechanism by which the chromosome 5q33.3 locus might promote human longevity, we analyzed the cause-specific mortality of rs2149954. Carriers of the minor allele of rs2149954 have a lower mortality risk for cardiovascu- lar disease (CVD) (HR ¼ 0.86, P ¼ 0.004), which mainly appeared to be caused by protection from stroke (HR ¼ 0.60,

Table 1. Results of the discovery phase, replication phase and joint analysis of cases aged ≥85