Table 3

Longevity association statistics for the combined analysis of the German and French samples.

dbSNP ID Gene Chromosome Alleles MAF controls Corresponding P P OR 95% CI P P P 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) 0.445 0.151 0.225 1.10 0.97–1.26 0.158 0.097 0.460

tional genes involved in various longevity pathways. Three of the tested 500 nsSNPs revealed a P ≤0.01. They are located in CCA three different genes: DUSP6, NALP1 and PERP. All of them are involved in the induction of apoptosis [41–43] and other diverse pathways linked to longevity or the aging process. The DUSP6 protein negatively regulates members of the mitogen-activated protein (MAP) kinase superfamily (MAPK/ERK, SAPK/JNK, p38), which are associated with cellular proliferation and differenti- ation [44–46]. Additionally, down-regulation of DUSP6 protein expression enhances carcinogenesis [47–49]. Its function as a tumor-suppressor gene [48,50] renders DUSP6 a potential modifier of human longevity. Due to its involvement in apoptosis and cell cycle control [51], down-regulation of PERP has also been linked to different types of cancer [52–54]. Besides its role as a key media- tor of programmed cell death [42] NALP1 plays an important role in the immune system [55–56] and DNA sequence variants in the NALP1 region were shown to be associated with the risk of several epidemiologically associated autoimmune and autoinflammatory diseases [57]. Hence, DUSP6, NALP1 and PERP are very good can- didates for influencing human health and, thus, longevity. Genetic variants that are more frequent in the long-lived than in the control population (the T-allele of nsSNP rs2301582 [NALP1] and the G- allele of rs648802 [PERP]; Table 1) can be regarded as beneficial by enhancing tumor-suppressor activity (PERP) or strengthening the immune system (NALP1). In contrast, genetic variation that inter- feres with proper protein function could have detrimental effects on human health and would therefore be expected to be less fre- quent in population strata of increasing age, as already seen for APOE [6,17] and the alpha-2-macroglobolin gene (A2M) [58]. This may also be the case for the C-allele of rs770087 in the DUSP6 gene that is less frequent in the case population (Table 1) and could potentially promote carcinogenesis by down-regulation or distur- bance of DUSP6 protein expression. Although it is feasible that the three tested variants rs770087, rs2301582 and rs648802 influence humanlongevity,analysesinalargerFrenchreplicationsampledid not confirm our initial observations in Germans.

of the largest centenarian collections to-date. The French sample had a power of 80% to replicate an association with an OR of 1.5 (as observed for SNP rs770087 in Germans; Supplementary Fig. 1). However, with an OR of 1.3 (as for rs2301582 and rs648802 in Germans), a sample size of about 1000 cases (with a case–control- ratio of 1) would be required for 80% power (Supplementary Fig. 2). Hence, to identify candidate genes with modest effects and to overcome the well-known replication problems in association experiments, it may be necessary to increase the study power by enlarging samples. Therefore, large-scale international coopera- tions like the GEnetic of Healthy Aging (GEHA) project [66], which studies thousands of long-lived individuals, provide a promising approach for future analyses and replication experiments. Another strategy is to perform meta-analyses. On the other hand, the failed replication in the French might be due to a population-specific effect, as different genetic and environmental factors and possi- ble interactions might cause certain effects in specific populations [67–70].

With 343 genes investigated, our study presents one of the largest candidate studies in human longevity research to-date. Thus, the considerable number of analysis results (Supplementary Table)isdeemedavaluablereferencepointforfurthergeneticstud- ies and helps to provide a balanced view of all obtained research evidence.

Conflict of interest statement

None.

Acknowledgements

We thank all study participants for their cooperation. For excel- lent technical assistance, we wish to acknowledge the laboratory personnel of the Institute of Clinical Molecular Biology as well as the staff of the Biological Resource Center of the Fondation Jean Dausset-CEPH. The authors are grateful to the technical and logistic support and helpful discussions with Simone Günther and Andrea Toeppel from Applied Biosystems. This study was supported by (1) the German Federal Ministry of Science and Education (BMBF) through an Explorative Project of the National Genome Research Network (NGFN), (2) the DFG Excellence Cluster ‘Inflammation at Interfaces’ and (3) the French Ministère de l’Enseignement Supérieur et de la Recherche.