The genetic markers were subjected to allelic (CCA) and genotypic (CCG) case–control comparisons. The cSNP with the lowest P , rs735943, was located in exon 10 of the EXO1 gene CCA (P = 8.16  10(cid:3)4 and P = 0.0012; Table 1). An additional CCA CCG association signal (rs4149965) was observed in exon 11 of the same gene (Table 1). Two other cSNPs located in different genes also showed nominally significant association with longevity, but since their P and P were much higher than those for CCA CCG the EXO1 cSNP rs735943 (Table S2), they were not studied any further. The P value of the EXO1 lead SNP rs735943 did not CCA pass correction for multiple testing (Bonferroni-adjusted P = 0.075; 92 tests). But as rs735943 was identified as the CCA top-ranking marker and was additionally supported by a second signal in a neighbouring exon, we performed an in-depth exploration of the EXO1 gene. This included mutation detection, fine mapping and functional studies to identify and verify the mechanistically relevant polymorphism(s) that would even- tually be subjected to genetic replication (Fig. 1A). Sequencing of all known exons, exon–intron boundaries and the promoter of EXO1 in 46 centenarians revealed several known and one novel mutation. In the subsequent fine mapping, tagging SNPs were considered as well as polymorphisms derived from the re- sequencing (i.e. cSNPs, SNPs located in the 50-UTR and further upstream region), using a total of 43 markers in the gene region (Table S3). Fourteen of these SNPs were significantly associated (Fig. 1B), and seven of these were located in potentially functional regions (Table 2 and Fig. 1C). The seven SNPs were in moderate to strong linkage disequilibrium (LD) with each other (Fig. 1B). When analyzed separately by logistic regression with adjustment for the apolipoprotein E gene (APOE), the only gene shown to have a large effect on longevity (OR for APOE see below), all of the seven SNPs exhibited a stronger effect than without adjustment (Table 2). No significant interaction was observed between any two SNPs or between a SNP and APOE. As expected, APOE alone showed a very strong association

Analysis of transcription factor binding sites

For analysis of EXO1 promoter regions containing the allelic variants at the SNPs thesequences wereanalyzedforpotentialtranscription binding sites using JASPAR (http://jaspar.genereg.net/).

Preparation of nuclear extracts from human cell lines and bandshift assays were performed as previously described (Rosenstiel et al., 2003). The sequence of the oligonucleotides representing the allelic variants of the EXO1 promoter were EXO1P_rs1776180_C:50-CGGGCCGGCAGCAGCGGCTGCAGTCGTATGGCAGTGAGCC- GCTGTCTACC-30 and EXO1P_rs1776180_G: 50-CGGGCCGGCAGCAGCGGCTGGA- GTCGTATGGCAGTGAGCCGCTGTCTACC-30 (the affected nucleotide position is underlined). For detection of E47 protein in DNA-binding complexes, bandshift assays were performed as described, and DNA-bound protein complexes were blotted onto PVDF membrane and analyzed for E47 protein using specific anti-E47 antibody (‘Shift Western’).

To analyze the tissue expression pattern of EXO1 and E47/TCF3 we and standard PCR techniques. Detailed materials and methods information can be found in

3. Results

3.1. Investigation of cSNPs in DNA repair genes reveals EXO1 as a candidate gene

We tested 92 non-synonymous coding SNPs (cSNPs) in 49 selected DNA repair genes for a possible association with the longevity phenotype (Table S1). These SNPs represent the ‘DNA repair subset’ of a large panel of 19779 cSNPs described in detail elsewhere (Hampe et al., 2007). In our experiment, we only typed this ‘DNA repair subset’ using the SNPlexTM Genotyping System

Table 1

Longevity association statistics for two coding SNPs in EXO1 (German

dbSNP ID MAF F11 F12 Corresponding Corresponding Corresponding P P CCA CCG controls controls controls AF centenarians F11 centenarians F12 centenarians rs735943 0.415 0.183 0.463 0.499 0.233 0.532 8.16(cid:4)10(cid:3)4 0.0012 rs4149965 0.279 0.073 0.411 0.229 0.061 0.336 0.0250 0.0504

MAF,minorallelefrequency;F11,frequencyofhomozygotesofminorallele;F12,frequencyofheterozygotes;AF,allelefrequency;P ,Pvalueobtainedfromanallele-based CCA case–control comparison, using a x2 test with 1 degree of freedom (df); P , P value obtained from a genotype-based case–control comparison, using a x2 test with 2df. CCG

Table 2

Longevity-associated EXO1 SNPs of potentially functional relevance (German

No. dbSNP ID Location Alleles MAF controls Corresponding AF P P P OR 95% CI CCA CCG APOE APOE (n=411) centenarians (n=395) 1 rs1776181 Promoter A/G 0.483 (A) 0.545 0.0133 0.0449 0.0095 1.31 1.07–1.62 2 rs1776180 Promoter C/G 0.430 (C) 0.507 0.0024 0.0068 0.0016 1.39 1.14–1.72 3 rs1776178 50-UTR A/G 0.412 (A) 0.488 0.0022 0.0050 0.0019 1.39 1.12–1.72 4 rs1635517 50-UTR G/A 0.425 (G) 0.507 0.0012 0.0033 9.82x10(cid:3)4 1.41 1.15–1.75 5 rs1776177 50-UTR C/T 0.472 (T) 0.400 0.0041 0.0133 0.0021 0.72 0.59–0.89 6 rs735943 Exon 10 A/G 0.415 (A) 0.499 8.16(cid:4)10(cid:3)4 0.0012 4.36(cid:4)10(cid:3)4 1.45 1.18–1.79 7 rs4149965 Exon 11 G/A 0.279 (A) 0.229 0.0250 0.0504 0.0118 0.74 0.58–0.93

MAF, minor allele frequency; n, number of analyzed individuals; P , P , see legend to Table 1; P , P value obtained by logistic regression analysis with adjustment for CCA CCG APOE APOE, using an additive model for the genotype–phenotype relationship of the respective EXO1 SNP; OR , odds ratio (per copy of the minor allele among controls) for APOE attaining old age, adjusted for APOE; 95% CI, 95% confidence interval for OR . APOE

Table 3

Subgroup analysis (females only) of EXO1 SNPs (German sample).