The genes of interest were derived from extensive literature research and from the Panther Classification System (http://panther.appliedbiosystems.com/navigation.jsp). They belong to longevity pathways (IIS and energy metabolism, apoptosis, cell cycle control, stress response, and intracellular signaling) or are based on genetic perturbations in animal models and on human diseases. The investigated nsSNP-assays were obtained from Applied Biosystems (Foster City, USA) and represent a subset of a large panel of 19779 nsSNPs described elsewhere [34] (http://marketing.appliedbiosystems.com/mk/get/GAAS HUMAN SNPGENO#). Altogether, we investigated 500 nsSNPs in 343 candidate genes (Supplementary Table).

Genotyping of the German samples was performed with the SNPlexTM Genotyping System (Applied Biosystems, Foster City, USA) [35]. The typing in the French replication sample was carried out with TaqMan SNP Genotyping Assays (Applied Biosystems).

3. Results 

The three top-ranking markers with respect to P (rs770087, CCA rs2301582 and rs648802) were located in three different genes [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 each showed 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 perform 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 discovery 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 genotyped 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), neither in the entire sample nor in the sex-specific analysis (data not shown). Also, the combined investigation of both Germans and French did not yield more significant values than for Germans alone (Table 3). The French study had a power of 50-80% to replicate the German findings (80% for rs770087 assuming an OR of 1.5 in Germans 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).

4. Discussion 

Previously, we have identified a polymorphism in the EXO1 gene as a likely modulator of human longevity in a targeted nsSNP approach [29]. Such an experiment is rapid and cost-efficient as it allows the direct evaluation of possible functional variants. Here, we applied the same strategy for the investigation of additional genes for association with longevity. 

In our analysis of the three candidate SNPs in the German and French samples, we also adjusted for APOE, the only gene shown to have a large effect (OR>2) on longevity [17,19], to reduce possible bias and confounding as well as to generate better estimates of the ORs. The results differed only negligibly from the non-adjusted ones (data not shown).

Table 1a 

Longevity association statistics for three nsSNPs (German sample). 

dbSNP ID Gene Chromosome Alleles MAF controls Corresponding P P OR 95% CI P CCA CCG additive n=410 AF centenarians n=386 rs770087 DUSP6 12 A/C 0Table 1b: Subgroup analysis (females only) for three nsSNPs (German sample). dbSNP ID Gene Chromosome Alleles MAF controls Corresponding P value OR 95% CI P value P value P value CCA CCG additive dom rez 1 dom rez 2 n=332 AF centenarians n=304 rs770087 DUSP6 12 A/C 0.204 (C) 0.151 0.013 0.040 1.45 1.08-1.92 0.015 0.059 0.036 rs2301582 NALP1 17 C/T 0.389 (T) 0.453 0.021 0.046 1.30 1.04-1.63 0.024 0.039 0.153 rs648802 PERP 6 C/G 0.432 (G) 0.513 0.004 0.019 1.39 1.11-1.73 0.005 0.037 0.012

Table2: Longevity association statistics for replication in French sample.dbSNP ID Gene Chromosome Alleles MAF controls Corresponding P value OR CCA CCG n=469 AF centenarians n=541 rs770087 DUSP6 12 A/C 0.184 (C) 0.193 0.599 0.622 1.06 rs2301582 NALP1 17 C/T 0.353 (T) 0.368 0.486 0.762  	1 .07 rs648802 PERP  	6 C/G  	0 .413 (G)  	041  	00  	0891  	0839  	101

Table 3: Longevity association statistics for the combined analysis of the German and French samples. dbSNP ID Gene Chromosome Alleles MAF controls Corresponding P value OR 95% CI P value P value P value CCA CCG additive dom rez 1 dom rez 2 n=879 AF centenarians n=927 rs770087 DUSP6 12 A/C 0.194 (C) 0.173 0.112 0.245 1.16 0.98-1.37 0.102 0.323 0.128 rs2301582 NALP1 17 C/T 0.368 (T) 0.402 0.037 0.053 1.16 1.01-1.33 0.037 0.019 0.212 rs648802 PERP  	6 C/G  	0 .421 (G)  	0445  	0151  	0225  	1 .10  	097  	0460