were tested for Hardy–Weinberg equilibrium in controls before inclusion in the analyses (P HWE >0.01). The genomic control analysis was conducted using PLINK V1.01 [37] (http://pngu.mgh.harvard.edu/purcell/plink/). Power and sam- ple sizes were calculated with the PS Power and Sample Size Program [38] (http://biostat.mc.vanderbilt.edu/twiki/bin/view/Main/PowerSampleSize) apply- ingasignificancelevelof0.05.Logisticregressionanalysiswasperformedtoanalyse thetwodifferentdominant–recessivemodels(firstalleledominantorsecondallele dominant) and an additive effects model on the logistic scale (multiplicativity of the odds ratios [OR]). In an additional analysis,APOE was included in the regression toadjustforitseffectonlongevity.Thenumberofε4alleleswastakenintoaccount for APOE and an additive allelic effect on the logistic scale was assumed for the three candidate genes. Possible interactions were investigated. For the adjustment of multiple testing, the classical Bonferroni method and the false discovery rate of Benjamini et al. [39] were applied. The regression analysis and the adjustment for multiple testing were performed with the statistics software program R (version 2.5.1) [40]. A p-value smaller than 0.05 was considered statistically significant.

Recently, we have identified exonuclease 1 (EXO1) as a candi- date for a novel longevity-enabling gene in a targeted approach by analyzing 92 nonsynonymous single nucleotide polymorphisms (nsSNPs) in 49 DNA repair genes [29]. Nonsynonymous SNPs result in amino acid changes and are likely to affect the structure and function of the produced protein. Together with SNPs in regulatory regionstheyaresupposedtohavethehighestimpactonthepheno- type [30]. In the present study, we have applied the same targeted nsSNP approach for the investigation of additional longevity path- ways(IISandenergymetabolism,intracellularsignalling,apoptosis and stress response) and genes in which mutations lead to genetic perturbations in animal models or to human diseases. Altogether, we tested 500 nsSNPs in 343 candidate genes for association with thelongevityphenotypeinaGermansamplecomprisingabout400 centenarians and an equal number of younger control subjects.

3. Results

In this investigation, 386 German centenarians (100–110 years) were compared to 410 appropriately matched younger controls (60–75 years) using an established longevity study design [31]. In 343 candidate genes, we typed 500 nsSNPs that represent a sub- set of a large panel described in detail elsewhere [34]. Gender- and region-matchedcase–controlanalyseswereperformedattheallele (CCA) and genotype (CCG) levels (Supplementary Table).

2. Material and methods

The three top-ranking markers with respect to P (rs770087, CCA rs2301582andrs648802)werelocatedinthreedifferentgenes[the dual specificity phosphatase 6 (DUSP6, GenBank No. BC037236), the NACHT leucine-rich-repeat protein 1 (NALP1, GenBank No. AB023143) and the p53 apoptosis effector related to PMP22 (PERP, GenBank No. AF317550)] and showed each a P ≤0.01 in the CCA entire sample (Table 1a). A subgroup analysis yielded similar p- values and frequency distributions for females (Table 1b). Because of the limited number of male centenarians (n=82) we did not per- form a male-specific analysis. None of the significant P values CCA of the three SNPs remained significant when adjusted for multiple testing by the conservative Bonferroni method or the false discov- ery method (Tables 1a and 1b). As the screening represented the explorative stage of this study and only served to identify markers for genetic replication, the three SNPs were subsequently geno- typed in an independent French collection of 541 centenarians and 469 younger controls. However, the genetic association observed in Germans could not be confirmed in the French (Table 2), nei- ther in the entire sample nor in the sex-specific analysis (data not shown). Also the combined investigation of both Germans and FrenchdidnotyieldmoresignificantvaluesthanforGermansalone (Table 3). The French study had a power of 50–80% to replicate the Germanfindings(80%forrs770087assuminganORof1.5asinGer- mans and 50% for rs2301582 and rs648802 each with an OR of 1.3 [Supplementary Fig. 1]). A sample size of about 1000 cases (with a case–control-ratio of 1) would be required to gain 80% power for the replication of SNP rs2301582 and rs648802 (Supplementary Fig. 2).

2.1. Study participants