In a few studies, such as the Ashkenazi Jewish Centenarian Study and the Leiden Longevity Study (LLS), different generations of long-lived families are being investigated for parameters and pathways contributing to the longevity phenotype (Atzmon et al., 2004; Schoenmaker et al., 2006). The survival benefit of the LLS families is marked by a 30% decreased mortality risk in the survival analysis of three generations, i.e., the parents of the probands in this study (nonagenarian sibling pairs), their unselected additional siblings, and their offspring (Schoenmaker et al., 2006). As compared to their partners, the offspring of Rs2075650 on chromosome 19 was the only SNP that was associated with survival into old age at the genome-wide significance level (P = 3.39 x 10^-17) (Table S2A). The minor allele of SNPs Study sample Analysis LLS GWAS: Cochran-Armitage trend test: GWAS 517K 403 90+ cases 1670 younger controls P < 1 x 10^-4 trend RS replication study: Meta-analysis: 960 90+ cases LLS GWAS 1825 younger controls RS replication study Leiden 85-plus replication study Leiden 85-plus replication study: Danish replication study 1208 85+ cases Replication 58 2090 younger controls Total: 4149 85+ cases Danish replication study: 7582 younger controls 1578 92+ cases 1997 younger controls P < 5 x10^-8 meta Leiden Longevity Study: 944 90+ cases Prospective analysis: Leiden 85-plus study: Follow-up 1,976 85+ cases All-cause mortality Danish 1905 cohort: 1578 92+ cases Fig.1 Flow chart of experimental work. Table1 Characteristics of the genotyped samples used for analysis. Study SNPs Samples Number Mean age Age range Men/women LLS GWAS 517K Cases Controls RS replication study Leiden Longevity Study; RS, Rotterdam Study; GWAS, genome-wide association study. showed an association with survival, with a higher prevalence of the minor allele in nonagenarians [OR = 1.31 (95% CI 1.17-1.46), P = 1.35 x 10^-6].

The remaining 57 SNPs did not show genome-wide significant effects on longevity either in men or women (Table S2B for men and S2C for women). The association of rs2075650 with survival did show some heterogeneity across the four studies (P = 0.0495), which is mainly because of the RS. We observed only moderate linkage disequilibrium (LD) between rs2075650 and rs429358 (r^2 = 0.553) and low LD between rs2075650 and rs7412 (r^2 = 0.014) when analyzing all samples with genotype data of rs2075650, rs429358, and rs7412 (n = 8946). Nevertheless, in a conditional analysis with rs429358 and rs7412 (Model 1, described in the Experimental procedures section), rs2075650 was no longer associated with longevity [OR =Present in the AlzGene database [http://www.alzgene.org/, (Bertram et al., 2007)], we analyzed the effect of 751 measured Alzheimer's disease SNPs on survival into old age in the LLS GWAS. Apart from rs2075650, none of the SNPs showed a significant association after adjustment for multiple testing (Table S4). Analysis of FOXO3A and AKT1 SNPs revealed that, apart from APOE, two other genes have shown consistent evidence for association with longevity, FOXO3A (Willcox et al., 2008; Flachsbart et al., 2009; Pawlikowska et al., 2009; Soerensen 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 association 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, we performed GWAS analysis of 403 nonagenarian cases and 1670 population controls. Of the 62 top associating SNPs, 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 generations, 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 longevity (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 (r^2 = 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 Leiden 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 independent of rs429358, which indicates that the association of rs2075650 with longevity is most likely due to variation in the APOE gene.