Because the longevity phenotype is inherited in the LLS fami- lies, they offer a route to identify genetic variants that influence human longevity. Previously, we tested whether the absence of GWAS-identified alleles promoting common diseases might explain their familial longevity (Beekman et al., 2010). Longevity was not easily explained by the absence of disease-susceptibility alleles. More likely therefore, the genome of the long-lived har- bors longevity-promoting alleles. To identify such loci, we per- formed a GWAS comparing nonagenarian siblings from the LLS and younger population controls. We subsequently investigated emerging candidate SNPs in nonagenarian cases from the Rot- terdam Study, the Leiden 85-plus study, and the Danish 1905 cohort.

In most experimentally modified animal model systems, sin- gle-gene mutations in many different genes have major life extension effects (Fontana et al., 2010; Kenyon, 2010). How- ever, natural human and animal longevity is presumed to be a complex trait (Finch & Tanzi, 1997). In humans, both candidate gene and genome-wide genetic association approaches have been applied in an attempt to identify longevity loci. The fre- quency of genetic variants has been typically compared between nonagenarian cases and young controls, revealing loci at which genetic variants may contribute to a higher or lower probability of survival into old age. The initial candidate gene studies aimed at finding human longevity genes were dominated by contradictory results (Christensen et al., 2006). The more consistent evidence obtained by repeated observa- tion in independent cohort studies for association with longev- ity has so far only been observed for three loci, the apolipoprotein E (APOE) locus (Schachter et al., 1994; Chris- tensen et al., 2006), the FOXO3A locus (Willcox et al., 2008; Flachsbart et al., 2009; Pawlikowska et al., 2009; Soerensen et al., 2010), and the AKT1 locus (Pawlikowska et al., 2009). Thus, despite the expectation that longevity would be influ- enced by many genetic variants with small effect sizes, the effect of variants has consistently been shown in only three genes.

Results

GWAS

A GWAS was performed in nonagenarian participants from the LLS and middle-aged controls from the Rotterdam Study (RS). Genotype data for 516,721 SNPs that passed quality control thresholds were analyzed in a comparison of 403 unrelated nonagenarians (94 years on average) and 1670 controls (58 years on average). A flow chart of the consecutive analysis steps is depicted in Fig. 1, and a description of the population samples investigated in the GWAS and subsequent replication studies is given in Table 1. Results of the association analysis of stage 1 are depicted in Fig. S1. None of the SNPs reached genome-wide significance (P < 5 · 10)8).

Hypothesis-free genome-wide approaches have also been undertaken. Genome-wide linkage scans reported evidence for linkage with longevity on chromosome 4q25 (Puca et al., 2001), 3p24-22, 9q31-34, and 12q24 (Boyden & Kunkel, 2010). How- ever, the evidence for these loci is still very weak as the results, obtained in centenarians and their families, could not be repli- cated in nonagenarian sibling pairs (Beekman et al., 2006) or have yet to be tested in other studies. A meta GWAS of survival to 90 years or older in 1836 cases and 1955 controls did not find any significant genome-wide associations (Newman et al., 2010). Thus far, hypothesis-free approaches have not identified any loci involved in longevity.

Replication studies

We prioritized the SNPs that had the most significant association with survival into old age according to the analysis of stage 1 (P < 1 · 10)4, Table S1). For 58 of the 62 selected SNPs, suc- cessful genotyping was obtained in the replication cohorts. In stage 2, these 58 SNPs were tested for association comparing 960 RS replication cases (mean age of 93 years), 1208 Leiden 85-plus replication cases (mean age of 92 years), and 1578 Dan- ish replication cases (mean age of 93 years) with appropriate middle-aged population controls (Table 1). Meta-analysis for the 58 SNPs, comprising a total of 4149 nonagenarian cases and 7582 younger controls (from the LLS GWAS, RS replication, Leiden 85-plus replication, and Danish replication studies), was performed.

In a few studies, such as the Ashkenazi Jewish Centenarian Study and the Leiden Longevity Study (LLS), different genera- tions 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 unse- lected 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 signi- ficance level (P = 3.39 · 10)17) (Table S2A). The minor allele

Nr of SNPs Study sample Analysis LLS GWAS: Cochran-armitage trend test: GWAS 517K 403 90+ cases 1670 younger controls P < 1 × 10–4 trend RS replication study: Meta-analysis: 960 90+ cases LLS GWAS 1825 younger controls RS replication study 85-plus replication study 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 × 10–8 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.

Table 1 Characteristics of the genotyped samples used for analysis