3.4. APOE

The array-based genotypes of SNP rs4420638 were confirmed 1 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 the latter (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 (r2 = 0.72, D0 = 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 as this is the sample in which the APOE–longevity association was originally detected (Scha¨chter et al., 1994).

3.2. Stage 2 analyses

The 16 SNPs selected for follow-up were next genotyped in an additional sample of 754 German LLI (mean age: 96.9 years) and 860 controls (mean age: 67.3 years; stage 2; Table 1), matched for ancestry, gender and geographical origin within Germany. This sample had a nominal power between 83% and 99% to detect the initially observed associations at the 5% significance level (with Bonferroni correction, the power was between 51% and 93%). However, only the association between longevity and rs4420638 was replicated (P = 1.1   10(cid:3)8, Bonferroni adjusted CCA P = 1.3 Sx  10(cid:3)7, Table 3), yielding a P value <2.2 Lx 10(cid:3)16 in CCA CCA the combined analysis of stage 1 and stage 2.

3.5. FOXO3A and EXO1

The recently confirmed longevity gene FOXO3A (Anselmi et 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 compara- tively high P values of 0.007 and 0.035, respectively, and were CCA 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 CCA

  -log10(P)    1aa 1me Chr1 me Chr2 emm Chr3 cee Chr4 meme ChrS meme Chr6 mem Chr? eee Chr8 eee ChrS meme Chri0 eee Chri1 sme Chri2 em Chris meme Chri4 mmm Chris:Ga a a nena re ae 

Fig. 2.‘‘Manhattanplot’’.CalculatedforallSNPsthat passedqualitycriteriaandshowedgoodcluster plots(clusterplotswereonlyinspected uptoaP value(cid:5)9.6   10(cid:3)6). min 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 couldnotbereplicatedinstage2.TheplotwascreatedusingHaploview4.2andallelicPvalues.(Forinterpretationofthereferencestocolorinthisfigurelegend,thereaderis 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 P OR 95% C.I. Bonferroni GC CAT CCA CCG (controls) adjusted P CCA 1 175309154 rs12741354 0.451 0.527 7.4(cid:2)10(cid:3)6 8.0(cid:2)10(cid:3)6 4.3(cid:2)10(cid:3)5 0.74 0.65–0.84 1 2 28515768 rs2338013 0.194 0.264 7.4(cid:2)10(cid:3)7 8.9(cid:2)10(cid:3)7 3.4(cid:2)10(cid:3)6 0.67 0.58–0.79 0.591 5 52022995 rs350450 0.219 0.287 4.2(cid:2)10(cid:3)6 3.2(cid:2)10(cid:3)6 2.2(cid:2)10(cid:3)5 0.69 0.59–0.80 1 6 29731718 rs29228 0.176 0.238 4.7(cid:2)10(cid:3)6 4.5(cid:2)10(cid:3)6 2.5(cid:2)10(cid:3)5 0.68 0.58–0.80 1 6 29753592 rs3129063 0.175 0.240 2.2(cid:2)10(cid:3)6 2.1(cid:2)10(cid:3)6 1.1(cid:2)10(cid:3)5 0.67 0.57–0.79 1 6 29778631 rs3129046 0.200 0.276 1.2(cid:2)10(cid:3)7 8.0(cid:2)10(cid:3)8 6.4(cid:2)10(cid:3)7 0.66 0.56–0.77 0.053 6 29785931 rs1610742 0.200 0.276 1.6(cid:2)10(cid:3)7 1.2(cid:2)10(cid:3)7 8.7(cid:2)10(cid:3)7 0.66 0.56–0.77 0.079 6 29808162 rs1610601 0.200 0.270 1.1(cid:2)10(cid:3)6 1.0(cid:2)10(cid:3)6 6.0(cid:2)10(cid:3)6 0.68 0.58–0.79 0.664 6 29834025 rs1633063 0.202 0.270 2.3(cid:2)10(cid:3)6 1.8(cid:2)10(cid:3)6 1.1(cid:2)10(cid:3)5 0.68 0.58–0.80 1 9 10715294 rs11790055 0.375 0.305 7.1(cid:2)10(cid:3)6 9.4(cid:2)10(cid:3)6 2.1(cid:2)10(cid:3)5 1.38 1.20–1.58 1 9 10726580 rs10959258 0.374 0.304 9.1(cid:2)10(cid:3)6 1.3(cid:2)10(cid:3)5 3.1(cid:2)10(cid:3)5 1.36 1.19–1.57 1 13 46905819 rs9595687 0.036 0.069 8.0(cid:2)10(cid:3)6 9.5(cid:2)10(cid:3)6 1.1(cid:2)10(cid:3)5 0.49 0.36–0.68 1 13 47001519 rs1575892 0.032 0.066 4.5(cid:2)10(cid:3)6 3.7(cid:2)10(cid:3)6 1.1(cid:2)10(cid:3)6 0.45 0.32–0.63 1 16 73418057 rs16947526 0.056 0.097 6.3(cid:2)10(cid:3)6 5.0(cid:2)10(cid:3)6 1.8(cid:2)10(cid:3)5 0.56 0.43–0.72 1 18 54052953 rs158869 0.484 0.406 2.3(cid:2)10(cid:3)6 3.0(cid:2)10(cid:3)6 1.8(cid:2)10(cid:3)6 1.37 1.20–1.56 1 19 50114786 rs4420638 0.109 0.185 3.7(cid:2)10(cid:3)10 1.8(cid:2)10(cid:3)10 1.6(cid:2)10(cid:3)10 0.53 0.44–0.65 1.2(cid:2)10(cid:3)4

A. Nebel et al. / Mechanisms of Ageing and Development 132 (2011) 324–330

Chr.:chromosome;LLI:long-livedindividuals;MAF(LLI):minorallelefrequencyinLLI;AF(controls):frequencyincontrolsoftheminoralleleinLLI;P CAT anallele-basedcase–controlcomparison,usingFisher’sexacttest;P CCG the major allele in LLI as reference; 95% C.I.: confidence interval (95%) of the OR; Bonferroni adjustment was made for 664,472 tests; GC P value: P adjusted for genomic control (l: 1.06). CCA

Table 3

Stage 2 validation of the 16 most significantly associated SNPs from the GWAS (stage 1).