genes for association with the longevity phenotype in a German sample comprising approximately 400 centenarians and an equal number of younger control subjects. This study is one of the largest candidate studies in human genetic longevity research to date. The study investigated genes involved in longevity pathways, such as insulin receptor/insulin-like growth factor-I signaling and energy metabolism, intracellular signaling, apoptosis, and stress response. Additionally, the study looked at genes in which mutations lead to genetic perturbations in animal models or human diseases. The top-ranking markers located in the genes DUSP6, NALP1, and PERP revealed p-values below 0.01 in allelic case-control comparisons. The association signals in Germans were not replicated in an independent French sample; however, the large number of analysis results is deemed a valuable reference point for further genetic studies.

Longevity in humans is considered a multifactorial trait to which both genetic and environmental factors contribute. Epidemiological studies have shown that the modulation of aging processes is inherited in long-lived families, and genetic factors have been estimated to account for about 25-32% of the variation in human adult lifespan. A twin study has revealed that genetic influences on survival are minimal before the age of 60 but increase thereafter. It is still debated to what extent a long and healthy life is based on the absence of harmful genetic variants that result in age-related disorders and/or on the presence of polymorphisms with beneficial effects in so-called longevity-enabling genes.

There is mounting evidence from experimental model organisms (S. cerevisiae, C. elegans, D. melanogaster, M. musculus) for the existence of core survival/anti-aging pathways that influence the aging process by increasing an organism's capacity to cope with stress-induced molecular damage and thus represent key candidates for attaining longevity. On the other hand, disruptions in these pathways lead to age-related failure of cellular maintenance and function, reduced stress tolerance, diseases, and ultimately death. Mutations in single genes involved in the pathways can either delay or accelerate the aging process in diverse model organisms. Many of the survival genes and pathways are evolutionarily conserved and also present in H. sapiens, thus providing a rationale to search for corresponding human loci. However, longevity candidate studies in humans are often hampered by difficulties replicating results in independent population samples. The most consistent association findings so far have been reported for the apolipoprotein E (APOE) and the forkhead box O3A (FOXO3A) genes.

The 500 nonsynonymous single nucleotide polymorphisms (SNPs) in 343 candidate genes 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 (http://pngu.mgh.harvard.edu/purcell/plink/). Power and sample sizes were calculated with the PS Power and Sample Size Program (http://biostat.mc.vanderbilt.edu/twiki/bin/view/Main/PowerSampleSize), applying a significance level of 0.05. Logistic regression analysis was performed to analyze the two different dominant/recessive models (first allele dominant or second allele 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 to adjust for its effect on longevity. The number of four alleles was taken into account 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. were applied. The regression analysis and the adjustment for multiple testing were performed with the statistics software program R (version 2.5.1). A p-value smaller than 0.05 was considered statistically significant.

Recently, exonuclease 1 (EXO1) was identified as a candidate for a novel longevity-enabling gene in a targeted approach by analyzing 92 nonsynonymous single nucleotide polymorphisms (nsSNPs) in 49 DNA repair genes. 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 regions, they are supposed to have the highest impact on the phenotype. In the present study, the same targeted nsSNP approach was applied for the investigation of additional longevity pathways (IIS and energy metabolism, intracellular signaling, apoptosis, and stress response) and genes in which mutations lead to genetic perturbations in animal models or human diseases. Altogether, 500 nsSNPs in 343 candidate genes were tested.Genes for association with the longevity phenotype in a German sample comprising about 400 centenarians and an equal number of younger control subjects were investigated. 

2. Material and methods 

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 subset of a large panel described in detail elsewhere [34]. Gender- and region-matched case-control analyses were performed at the allele (CCA) and genotype (CCG) levels (Supplementary Table).