showed a significant association after adjustment for multiple testing (Table S4).

selameFselaMllA 066.081.0ot11.0)30.03121611.040.0ot63.0)61.0)1201883.070.0ot71.0)50.0)4322)1)Llomm(esoculG 004.060.1ot78.069.03711851.030.1ot48.039.0099321.020.1ot88.059.03612–)1)LUm(nilusnI 682.020.0ot80.0)30.0)0121460.000.0ot90.0)40.0)8101260.000.0ot70.0)40.0)8222)1)Llomm(loretselohcLDH 438.070.1ot59.010.18021590.051.1ot99.070.16101302.080.1ot89.030.19222–)1)Llomm(edirecylgirT 561.020.0ot90.0)40.0)8021951.020.0ot01.0)40.0)1101160.000.0ot80.0)40.0)9122)mn(eziSLDH 642.030.0ot41.0)50.0)8021711.020.0ot91.0)80.0)1101260.000.0ot31.0)60.0)9122)mn(eziSLDL 074.031.0ot60.0)30.08021721.061.0ot20.0)70.05101141.021.0ot20.0)50.03222)1)Llomp(3Tf .eninoryhtodoiirteerf,3Tf;nietorpopilytisned-wol,LDL;nietorpopilytisned-hgih,LDH .sisylanaehtniselpmasforebmuN,N* .853924srfoelellaronimrepretemarapmuresnotceffE;tceffE†

Analysis of FOXO3A and AKT1 SNPs

Apart from APOE, two other genes have shown consistent evi- dence for association with longevity, FOXO3A (Willcox et al., 2008; Flachsbart et al., 2009; Pawlikowska et al., 2009; Soeren- sen et al., 2010) and AKT1 (Pawlikowska et al., 2009). For the longevity-promoting FOXO3A SNPs previously reported with centenarian longevity, we observed no association with survival into old age in our nonagenarians (Table S5). For AKT1, one of the two measured SNPs, rs2498804, showed a significant asso- ciation with survival into old age [OR = 0.75 (95% CI 0.63– 0.89), P = 0.001] (Table S5).

Discussion

To identify common SNPs contributing to longevity, GWAS analysis of 403 nonagenarian cases and 1670 population con- trols was performed. Of the 62 top associating SNPs, 58 were tested in a meta-analysis of 4149 nonagenarian cases and 7582 younger controls and we identified one SNP, rs2075650, that associated significantly with survival into old age (P = 3.39 · 10)17). Carriers of the minor allele had a 29% decreased probability of reaching 90 years on average. Although cases and controls originate from different genera- tions, we concluded that there was no substructure to an extent that would affect the observations.

Rs2075650 is located in the TOMM40 gene at chromosome 19q13.32 close to and centromeric of the APOE gene (Fig. S2), which has shown consistent evidence for association with lon- gevity (Schachter et al., 1994; Christensen et al., 2006). The ApoE protein has three isoforms (ApoE e2, ApoE e3, and ApoE e4) that are defined by two SNPs, rs7412 (Arg136Cys; e2) and rs429358 (Cys112Arg; e4). ApoE e4 carriers have an increased risk of cardiovascular disease and Alzheimer’s disease, while ApoE e2 carriers are protected from these diseases (Corder et al., 1993; Eichner et al., 1993; Christensen et al., 2006). Although we detected only moderate LD (r2 = 0.553) between rs2075650 and the ApoE e4-defining SNP rs429358, we could not detect a significant effect of rs2075650 on longevity independent of rs429358. Several prospective studies, including one with the Danish 1905 cohort (Jacobsen et al., 2010), reported increased mortality for ApoE e4 carriers, even though there is still much debate about APOE being a ‘frailty gene’ or a ‘longevity gene’ (Gerdes et al., 2000; Christensen et al., 2006; Ewbank, 2007; Jacobsen et al., 2010). The prospective data in the LLS and Lei- den 85-plus study support the ‘frailty gene’ hypothesis, as rs429358 affects mortality after 85 years and continues the effect after 90 years [HR = 1.08 (95% CI 1.03–1.13), P = 0.001 and HR = 1.08 (95% CI 1.03–1.12), P = 0.001, respectively] (Table S3B, Fig. S4). In these prospective studies, carriers of the minor allele of rs2075650 showed no increased mortality inde- pendent of rs429358, which indicates that the association of

rs2075650 with longevity is most likely due to variation in the APOE gene. Although GWAS studies have reported significant associations between rs2075650 and Alzheimer’s disease, brain imaging, total cholesterol, and CRP plasma levels (Reiner et al., 2008; Aulchenko et al., 2009; Seshadri et al., 2010; Shen et al., 2010), no analyses were performed to determine whether these associations are APOE independent. We observed no APOE- independent effect on the phenotypes investigated in the LLS offspring and partners except for total and LDL cholesterol.

The strength of this study is that, by using a GWAS, we were able to replicate the previously reported association of the APOE locus with longevity (Schachter et al., 1994; Christensen et al., 2006) as the major locus. This was not observed in the previously published meta genome-wide association study of Newman et al. (Newman et al., 2010), possibly because of differences in the study design and population control selection between the studies. While Newman et al. used nonagenarian cases in a pop- ulation-based design, we made use of a family-based design in which the families are genetically enriched for longevity. In addi- tion, Newman et al. used population controls from the same cohort which had died before the age of 80. Between 60 and 80 years however, there might already have been a selection on survival, decreasing the frequency of ApoE e4 carriers in the con- trol group. In contrast, we made comparisons to a younger pop- ulation group (55–60 years) from a different cohort (RS).