1 LLI 763 99.7 101 94-110 
1 Controls 1085 60.2 60 45-77 
2 LLI 754 96.9 97 95-108 
2 Controls 860 67.3 66.5 60-75 

LLI: long-lived individuals. 

Fig. 1 shows a workflow diagram summarizing the different stages of the study. The association did not withstand Bonferroni correction (Bonferroni-adjusted P = 0.091 assuming 13 tests; see Supplementary CCA methods). As SNP rs12741354 had the second lowest P value, it was also investigated in an independent collection of 536 French centenarians (mean age: 103.7 years) and 508 younger controls (mean age: 50.8 years) (power: 65.5%) where it failed to show any association with longevity (P = 0.86; Supplementary CCA Table 1). 

Sixteen SNPs were selected for follow-up and genotyped in stage 2 (Table 2). Their nominal P values ranged from 1.8 x 10^-10 to 9.5 x 10^-6. Due to multiple testing in GWAS, usually low initial P values in stage 1 are required to be able to replicate the association. Our follow-up study did not have sufficient power to detect SNP-effects in stage 2 with higher P values (>10^-5). Therefore, additional SNPs were not included in our follow-up. Only the association between longevity and SNP rs4420638 near the APOC1 gene achieved genome-wide significance (Bonferroni-adjusted P = 1.2 x 10^-4, based upon 664,472 tests and assuming no CCA correlation between SNPs; see Fig. 2 and Supplementary Fig. 4). The significance of the other 15 SNPs did not withstand Bonferroni correction, thereby highlighting the need for independent validation.

The array-based genotypes of SNP rs4420638 were confirmed by a TaqMan SNP Genotyping Assay (99.4% concordance). SNP rs4420638 is located 14 kb downstream of the APOE locus (Corder et al., 1993) and has previously been shown to be in strong linkage disequilibrium (LD) with it (Coon et al., 2007). Defining APOE e4 by intragenic SNP rs429358, we were able to confirm this LD in the German control sample of stage 2 (r^2 = 0.72, D' = 0.97). Logistic regression analysis including both rs4420638 genotype and APOE e4 status showed that the longevity association of rs4420638 was explicable by its LD with APOE (P = 0.57 for the association between longevity and rs4420638, after adjustment for e4 status). SNP rs4420638 was not further analyzed in the French sample as this is the sample in which the APOE-longevity association was originally detected (Schachter et al., 1994).

The 16 SNPs selected for follow-up were next genotyped in an additional sample of 754 German LLIet al., 2009; Flachsbart et al., 2009; Li et al., 2009; Pawlikowska et al., 2009; Soerensen et al., 2010; Willcox et al., 2008) and the longevity candidate EXO1 (Nebel et al., 2009) yielded comparatively high P values of 0.007 and 0.035, respectively, and were therefore far too large to qualify for follow-up in stage 2. Another SNP, rs12741354 in the astrotactin 1 (ASTN1) gene, yielded a nominally significant P value of 0.007, but this was too large to qualify for follow-up in stage 2. 

```
Chr1 me 
Chr2 emm 
Chr3 cee 
Chr4 meme 
Chr5 meme 
Chr6 mem 
Chr7 eee 
Chr8 eee 
Chr9 meme 
Chri0 eee 
Chri1 sme 
Chri2 emm 
Chri3 meme
Chri4 mmm
Chri5 Ga a nena re ae
```

Figure 2. Manhattan plot. Calculated for all SNPs that passed quality criteria and showed good cluster plots (cluster plots were only inspected up to a P value of 9.6 x 10^-6). Marker positions are in NCBI's build 36. Marker rs4420638 is the only SNP with genome-wide significance (red line). The large peak of significant SNPs on chromosome 6 could not be replicated in stage 2. The plot was created using Haploview4.2 and allelic P values. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Table 2. Summary statistics for the 16 SNPs most significantly associated with longevity in the GWAS (stage 1).

```
Chr. Position B36 ID MAF (LLI) AF P P OR 95% C.I. Bonferroni GC CAT CCA CCG (controls) adjusted P CCA
1 175309154 rs12741354 0.451 0.527 7.4x10^-6 8.0x10^-6 4.3x10^-5 0.74 0.65-0.84 1
2 28515768 rs2338013 0.194 0.264 7.4x10^-7 8.9x10^-7 3.4x10^-6 0.67 0.58-0.79 0.591
5 52022995 rs350450 0.219 0.287 4.2x10^-6 3.2x10^-6 2.2x10^-5 0.69 0.59-0.80 1
6 29731718 rs29228    0.176   0.238   4.7x10^-6   4.5x10^-6   2.5x10^-5   0.68   0.58-0.80   1
6    29753592    rs3129063    0.175    0.240    2.2x10^-6    2.1x10^-6   .1x10^-5    .67     .57-.79     l
6    '29778631    rs3129046    .200     .276      l.lx10^~7      a.OxlO^S      eAxlO^~7      .66     .56-.77     .053
6    29785931    rs1610742    .200     .276      1.6x10^-7      1.2x10^-7      8.7x10^-7      .66     .56-.77     .079
6    29808162    rs1610601    .200     .270      1.1x10^-6      l.OxlO^S      6.0x10^-6      .68     .58-.79     .664
6    29834025    rs1633063    .202     .270      2.3x10^-6      l.SxlO^S      l.lx10^-5     .68     .58-.80     l
9 10715294 rs11790055   0.375   0.305   7.1x10^-6   9.4x10^-6   2.1x10^-5   1.38   10.93, 0.78, 1.11, 1, 0.425, 6, 29753592, rs3129063, 0.202, 0.216, 0.353, 0.367, 0.528, 0.92, 0.77,
1.10, 1, 0.354, 6, 29778631, rs3129046, 0.237, 0.247, 0.523, 0.525, 0.744, 0.95, 0.80,
1.12, 1, 0.432, 6, 29785931, rs1610742, 0.234, 0.246, 0.454, 0.470, 0.742, 0.94, 0.79,
1.11, 1, 0.378, 6, 29808162, rs1610601, 0.225, 0.234, 0.548, 0.575 ,0 .734 ,095 ,080
1 .13 ,1 ,0549 ,6 ,29834025 ,rs1633063 ,0224 ,0240 ,0289 ,0303 ,0464 ,091 ,077
1 .08 ,1 ,0255 ,9 ,10715294 ,rs11790055 ,0328 ,0327 ,0942 ,0969 ,0876 ,101 ,086
1 .17 ,1 ,0929 ,9 ,10726580 ,rs10959258 ,0331 ,0329 ,0897 ,0895 ,0831 ,101 .087
1 .17 ,1 .0897 .13 .46905819 rs9595687 .053 .048 .470 .507 .957 1.13 .81
1 .56 ,1 .0488 .13 .47001519 rs1575892 .055 .049 .442 .461 .75 1.13 .82
1 .56 ,1 .0504 ,16 ,73418057 rs16947526 0077 ,0.087 ,0314 ,0354 ,0.628 ,088 ,068
1.14, 1, 0.338, 18, 54052953, rs158869, 0.450, 0.425, 0.169, 0.175, 0.308, 1.11, 0.96,
1.28, 1, 0.226, 19, 50114786, rs4420638, 0.104, 0.174, 1.9e10^08, 1.1e10^08, 8.8e10^08,
0.55, 0.44-0.68, 1.3e07