In response to CR, radiation or oxidative stress, increased SIRT1 activity has also been shown to reduce p53-, Bax- or forkhead-dependent apoptosis (Luo et al., 2001; Vaziri et al., 2001; Brunet et al., 2004; Cohen et al., 2004; Motta et al., 2004; Nemoto et al., 2004). A strong association has been reported between ageing and enhanced susceptibility to apoptosis (Higami and Shimokawa, 2000; Pollack et al., 2002). The SIRT1-mediated resistance to apoptosis may therefore promote long-term cell survival and mitigate the age-related progressive erosion of cells, tissues and organs. More recent findings in mouse embryonic fibroblasts have revealed an additional SIRT1 function in handling chronic cellular stress: the protein triggers replicative senescence (Chua et al., 2005). This role is in marked contrast to that reported for yeast cells where SIR2p promotes cell division. However, the ability of SIRT1 to induce permanent cell cycle arrest is only triggered by chronic, but not acute, genotoxic stress (Chua et al., 2005). Further work is needed to elucidate how the mammalian SIRT1 helps cells respond to adverse conditions and how this mechanism influences the ageing process.

2. Subjects and methods

2.1. Subjects

A total sample of 1026 unrelated German long-lived individuals (LLI) was studied. All individuals were between 95 and 109 years of age at the time of recruitment (mean age: 98.3 years). These included 386 centenarians and super- centenarians (mean age: 101.0 years). The gender ratio in the sample was 74% females vs. 26% males. The 547 German control subjects were between 60 and 75 years of age (mean age: 67.2 years) and matched the long-lived individuals by gender, ethnical ancestry and geographical origin within the country. A detailed description of the samples and the recruitment procedure is given in Nebel et al. (2005). All subjects gave informed, written consent prior to participation. The study was approved by the Ethics Committee of the University Hospital Schleswig-Holstein, Kiel and by the local data protection authorities.

The human SIR2 homologue SIRT3, which encodes a mitochondrial protein (Onyango et al., 2002), has previously been subject to a longevity association study in Italians from Calabria (Rose et al., 2003). By analyzing the genotype- specific survival function, an association between a silent mutation in the coding region of SIRT3 and survivorship was detected, albeit only in males. This finding suggests that variation in SIRT3 itself, or in a gene that is tightly linked to it, may modulate life expectancy in humans (Rose et al., 2003).

2.2. SNP genotyping

DNA samples from long-lived individuals and control subjects were analyzed for five SNPs located in the SIRT1 gene (Table 1) using the Taqman Allelic Discrimination method (Applied Biosystems) on an automated platform as previously described (Hampe et al., 2001a,b). Details about the assay designs are available from http://www.appliedbiosystems.com/. HapMap data (http://www.hapmap.org/) revealed that the entire SIRT1 gene is within a 151-kb region of strong linkage disequilibrium (an ‘LD block’). Such LD blocks are typically characterized by a few (2–4) common haplotypes that capture

The life-prolonging effects that the SIR2 gene and its homologues exert on various model organisms, together with the association finding in the human SIRT3 gene, prompted us to investigate whether common allelic variation in the SIRT1

Summary association statistics for the SIRT1 polymorphisms analyzed in German long-lived individuals and controls

SNP SNP location in MAFa LLIb Centenarians gene P c P d P e P f allele genotype allele genotype hCV3003909 (rs3758391) 50 region 0.323 0.93 0.99 0.53 0.82 hCV11642237 (rs1885472) Intron 0.325 1.00 0.98 0.57 0.82 hCV16179813 (rs2273773) Exon 5 (silent 0.067 0.71 0.82 0.69 0.80 mutation) hCV25611602 (rs10997870) Intron 0.340 0.88 0.95 0.62 0.85 hCV11642299 (rs2234975) 30UTR 0.082 0.64 0.42 0.53 0.27

c P value from a c2 test (1df) for allele frequency difference between LLI and controls. d P value from a c2 test (2df) for genotype frequency differences between LLI and controls. e P value from a c2 test (1df) for allele frequency difference between centenarians and controls. f P value from a c2 test (2df) for genotype frequency differences between centenarians and

1). Similarly, analysis with gender-stratified samples no evidence for association.

the majority (typicallyO90%) of the genetic variation present within the LD block (Daly et al., 2001). Consequently, by selecting five markers of varying minor allele frequency that are spread across the SIRT1 region we could be fairly confident of capturing the common haplotypes and the majority of the genetic variation.The tested polymorphisms are spacedacrossthe coding 0 region, the 3 UTR and the promoter of the gene (Table 1). By means of the ‘First Exon and Promoter Prediction Program for Human DNA’ (http://rulai.cshl.org/tools/FirstEF/) the promoter of the SIRT1 gene was localized within the genomic sequence NM_012238 to between about 500 and 1000 bp downstream of hCV3003909, i.e. the promoter lies between the two markers hCV3003909 and hCV11642237.