Fig. 1 Flow chart of experimental work.

Table 1 Characteristics of the genotyped samples used for analysis

Study SNPs Samples Number Mean age Age range Men⁄women LLS GWAS 517K Cases 403 94 89–102 137⁄266 517K Controls 1670 58 55–59 745⁄925 RS replication study 58 Cases 960 94 90–106 217⁄743 58 Controls 1825 62 60–65 805⁄1020 Leiden 85-plus replication study 58 Cases 1208 92 85–109 372⁄836 58 Controls 2090 35 15–70 743⁄1347 Danish replication study 58 Cases 1578 93 92–93 430⁄1148 58 Controls 1997 57 46–68 900⁄1097

Leiden Longevity Study; RS, Rotterdam Study; GWAS, genome-wide association study.

controls), showed a significant association of rs429358 with longevity [OR = 0.62 (95% CI 0.56–0.68), P = 1.33 · 10)23], which was comparable to rs2075650 [OR = 0.67 (95% CI 0.61– 0.74), P = 9.15 · 10)17]. Rs7412 also showed an association with longevity, with a higher prevalence of the minor allele in nonagenarians [OR = 1.31 (95% CI 1.17–1.46), P = 1.35 · 10)6].

was underrepresented among the older cases as compared to middle-aged controls, hence associated with the decreased probability of carriers surviving into old age corresponding to an odds ratio (OR) below unity [OR = 0.71 (95% CI 0.65-0.77)]. This effect is observed in both sexes (Table S2B, C). The remain- ing 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 (r2 = 0.553) and low LD between rs2075650 and rs7412 (r2 = 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 = 0.93 (95% CI 0.81–1.07), P = 0.337]. The OR increased from 0.67 to 0.93, i.e., the deleterious effect of rs2075650 on longevity diminishes and is statistically non-signif- icant. However, the deleterious effect of rs429358 [OR = 0.64 (95% CI 0.56–0.74), P = 2.68 · 10)9] and the protective effect of rs7412 [OR = 1.20 (95% CI 1.07–1.36), P = 0.002] on lon- gevity remained significant.

rs2075650 and the APOE e2⁄e3⁄e4 polymorphism

Rs2075650 is located in the TOMM40 gene, next to the APOE gene (Fig. S2). APOE was previously associated with longevity (Schachter et al., 1994; Christensen et al., 2006). The ApoE pro- tein has three isoforms (ApoE e2, ApoE e3, and ApoE e4) which are defined by two SNPs, rs7412 (Arg136Cys; e2) and rs429358 (Cys112Arg; e4). A meta-analysis of rs7412 and rs429358, in the LLS GWAS study, the Leiden 85-plus replication study, and the Danish replication study samples (3189 cases and 5757

To determine whether there was an APOE-independent effect of rs2075650 on survival after 90 years, prospective analysis of rs2075650, adjusted for rs429358 and rs7412, was performed. This analysis showed that carriers of the minor allele of rs2075650 displayed no increased mortality, i.e., a sig- nificant hazard ratio (HR) above 1, after 90 years of age inde- pendently of APOE in two of the three cohorts analyzed [LLS, HR = 0.99 (95% CI 0.78–1.25), P = 0.914; Leiden 85-plus study, HR = 1.06 (95% CI 0.89–1.27), P = 0.521; Danish 1905 cohort, HR = 1.21 (95% CI 1.01–1.44), P = 0.036) (Table S3A, Fig. S3).

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Overall, our results suggest that the association of rs2075650 with longevity is most likely a reflection of the effects of rs429358, caused by the moderate LD between the loci.

Association of rs429358 (e4) and rs2075650 with serum parameters

As previous studies showed that rs429358 was associated with several metabolic phenotypes (Boerwinkle & Utermann, 1988; Topic et al., 2008; Hubacek et al., 2010), association of this SNP with relevant serum parameters was determined in the off- spring of the elderly LLS cases and their partners (n = 2324, Model 2 described in the Experimental procedures section). We replicated the previously reported associations of rs429358 with plasma levels of ApoE (P = 7.42 · 10)28), total cholesterol (P = 0.001), LDL cholesterol (P = 4.91 · 10)5), HDL cholesterol (P = 0.062), and high sensitivity C-reactive protein (hsCRP) (P = 0.028) and with HDL (P = 0.061) and LDL particle size (P = 0.062) (Table 2). In addition, we detected a minor effect on IGF-1 (P = 0.025) and IGFBP3 levels (P = 0.042) (Table 2). The effect on IGF-1 seems to be female-specific (P = 0.005 and P = 0.748, in women and men, respectively) and is still signifi- cant after correction for multiple testing. We observed no APOE-independent effect of rs2075650 on these traits, except for an increase of 0.18 mmol L)1 total cholesterol (P = 0.017) and 0.14 mmol L)1 LDL cholesterol (P = 0.014) with each minor allele of rs2075650 (using Model 3 described in the Experimental procedures section).

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No significant effects of rs429358 were observed on glucose (P = 0.388), insulin (P = 0.123), triglyceride (P = 0.203), and fT3 (P = 0.141) levels (Table 3); the phenotypes that have previ- ously been associated, in middle age, with familial longevity in the LLS families (Rozing et al., 2009, 2010a,b; Vaarhorst et al., 2011; Wijsman et al., 2011).

Analysis of Alzheimer’s disease SNPs

Rs2075650 has consistently been associated with an increased risk of Alzheimer’s disease in several independent GWAS studies (Harold et al., 2009; Lambert et al., 2009; Seshadri et al., 2010). Therefore, we studied the effect of SNPs present in the AlzGene database [http://www.alzgene.org/, (Bertram et al., 2007)], on survival into old age in the LLS GWAS. Apart from rs2075650, none of the 751 measured Alzheimer’s disease SNPs