http://www.R-project.org/), adjusting for APOE. Interaction analysis of EXO1 SNP rs1776180 with the TCF3 SNPs was also performed by logistic regression. Furthermore, besides the additive model for the genotypes the two possible dominant-recessive models were also regarded. They yielded no significant improvement over the additive model and are thus not presented in our results. P values smaller than 0.05 were considered nominally statistically significant. If adjustmentformultipletestingwasconsidered,theBonferronimethod wasapplied.

2.5. Mutation detection

Sequencing of the exons, intron–exon boundaries and the promoter region of EXO1 was done in 46 centenarians with the BigDye1 Chemistry (Applied Biosystems) (for primer sequences, see Table S6). The EXO1promoter was localized

In the present study, we tested a total of 92 non-synonymous coding single nucleotide polymorphisms (cSNPs) in 49 DNA repair genes for a possible association with the advanced longevity phenotype. Our strategy was to derive a genetic association signal, to identify the mechanistically relevant polymorphism(s) and to subject these variants to genetic replication (Fig. 1A). We identified a candidate for a novel susceptibility locus for prolonged life expectancy, the gene encoding exonuclease 1 (EXO1). EXO1 participates in various DNA metabolic pathways affecting genomic stability, including homologous recombination and DNA damage repair (Liberti and Rasmussen, 2004; Qiu et al., 1999). Our detailed functional and genetic analyses revealed a common allele in the promoter of the gene that is enriched in female centenarians and that enhances EXO1 expression via differential binding of the transcription factor E47. Finally, the association between the functional SNP and longevity is replicated in an independent population of female French centenarians and controls.

  Identification of seven associated potentially functional EXO7 variants 

2. Materials and methods

2.1. Study participants

The German DNA collections and the recruitment procedures were reported in detail elsewhere (Nebel et al., 2005). The case sample comprised 395 centenarians (100–110yearsatascertainment;meanage:101.3years);79%ofthemwerefemale. Thecentenarianswerespecificallymatchedtoagroupof411youngercontrols(60–75 years;meanage:66.6years)intermsofancestry,genderandgeographicoriginwithin Germany (Nebel et al., 2005), thus minimizing any systematic genetic differences between these samples that might arise due to very low levels of undetected population structure. The French sample set consisted of 455 female centenarians (meanage:103.7years)and109femalecontrols(60–70years;meanage:64.5years) as described previously (Blanche´ et al., 2001). All subjects gave written informed consentpriortoparticipation.Thestudywasapprovedbytherespectiveinstitutional review committees of the participating centers.

2.2. Gene and cSNP selection

The investigated 92 coding SNPs (Table S1) are located in 49 genes involved (Hampe et al., 2007; http://cSNP.appliedbiosystems.com/, see link to SNPlexTM System cSNP Assay Index).

2.3. Genotyping

Fig.1.IdentificationofEXO1asacandidategeneforhumanlongevity.(A)Workflow diagram summarizing the different stages of the experiment. (B) Fine mapping of the EXO1 region on chromosome 1 (German sample). The physical position (in bases) of all genotyped SNPs with their allele-based P values ((cid:3)log P ) and a 10 CCA schematic representation of the gene structure for NM_130398 are shown. Coordinates refer to NCBI genome assembly build 36. The seven associated SNPs (markersindicatedbyanasterix(*);plottedassociationresultspresentedasablack circle (*)) wereconsidered tobeofpotentiallyfunctional importance astheywere locatedinthepromoter,the50-UTRorexons.TheLDplotofthelocusisbasedonthe measurer2andwasgeneratedwithHaploview3.32usingthegenotypesofGerman controlindividuals.(C)IllustrationoftheEXO1genemodelhighlightingtheposition ofthesevenpotentiallyfunctionalSNPs.EXO1hasseveralisoforms;herewereferto NM_130398 that contains 16 exons, 13 of which constitute the coding region (indicated by big boxes) whereas three exons (exon 1, exon 2 and alternative exon 2a) span the 50-UTR, and the last exon (nominal exon 15) contains coding sequence and the 30-UTR (small boxes). The seven SNPs analyzed in this study are located within functionally relevant regions: two SNPs in the 50-proximal promoter region (rs1776181A/G, rs1776180C/G), three SNPs in the 50-UTR (rs1776178A/G, rs1635517G/A, rs1776177C/T) and two non-synonymous SNPs in the coding region (rs735943A/G resulting in amino acid substitution H354R; rs4149965G/A resulting in V458M). ATG: start codon in nominal exon 3, TAA: stop codon in nominal exon 15. The position of the transcriptional start site as predicted by the Eponine prediction tool (http://www.sanger.ac.uk/Software/analysis/eponine/) is indicated by an asterisk (*).

Genotyping was performed using the SNPlexTM Genotyping System TaqMan1 SNP Genotyping Assays (Applied Biosystems, Foster City, USA). analyzed cSNPs had a minor allele frequency of at least 1%, a minimal overall (HWE) in controls (P > 0.05). HWE

2.4. Statistical analysis