As expected, the five SNPs comprised a single LD block with high levels of LD between all the markers. The pairwise 0 D values ranged from 0.92 to 1.00. The five SNPs defined five haplotypes with a frequencyO1% in the control population and with the three most frequent haplotypes accounting for 92% of the data (Table 2). Four of the markers were haplotype-tagging SNPs (htSNPs) in the sense that they were sufficient to resolve the five haplotypes. In other words, markers hCV3003909 and hCV11642237 were in perfect LD and redundant with respect to each other such that either could be used in conjunction with the other markers to reconstruct the haplotypes and their frequencies. None of the five-SNP haplotypes differed significantly in frequency between the case samples and the controls. Neither did testing of two-, three- and four-SNP haplotypes in a sliding window across the gene reveal any statistically significant frequency differences (data not shown).

2.3. Statistical analysis

Single marker case–control analyses on allele and genotype frequency data were performed using contingency table c2 statistics at the appropriate degrees of freedom using the Web- based Simple Interactive Statistical Analysis (SISA) tool, available from http://home.clara.net/sisa. The software program Haploview, version 3.2 (http://www.broad.mit.edu/mpg/haplo- view/) was used to assess all polymorphisms for significant deviation from the Hardy-Weinberg equilibrium, to calculate LD 0 (D ) between each marker pair and to visualize haplotype structure (Barrett et al., 2005). Haplotype frequencies were inferred using the expectation maximization (EM) algorithm and the sliding window option as implemented in the program COCAPHASE, version 2.403 which is part of the UNPHASED suite (http://portal.litbio.org/; Dudbridge, 2003). COCAPHASE was also used to evaluate the statistical significance of haplotype frequency differences between cases and controls.

4. Discussion

The SIR2/Sirt1 gene has been shown to regulate lifespan in many model organisms, including yeast, C. elegans and rodents (Hekimi and Guarente, 2003). In mammals, expression of the SIRT1 protein is activated by various stress stimuli, leading to a break-down of fat stores, induction of cell cycle arrest and DNA repair processes, or attenuation of apoptosis (Brunet et al., 2004; Cohen et al., 2004; Nemoto et al., 2004; Picard et al., 2004; Chua et al., 2005). Under adverse circumstances, SIRT1 appears to increase organismal longevity by tipping physiological responses away from cell death and towards cellular stress resistance (Brunet et al., 2004). As effective stress resistance is frequently correlated with extended lifespan in lower eukaryotes, the ability of SIRT1 to modulate reactions to stress in mammalian cells also suggests a potential link between the gene and longevity in mammals (Chua et al., 2005), and therefore possibly humans.

3. Results

The entire collection of 1026 German LLI and the sub-set of 386 centenarians were subjected to a gender-matched case–control analysis of five SNPs in the SIRT1 gene. All SNPs were found to be in Hardy-Weinberg equilibrium in both the cases and controls. No evidence for an association was detected between any of the tested SNPs and the longevity phenotype in either sample, neither at the allele nor at the genotype level

We therefore sought to determine whether common allelic variation in the SIRT1 gene was associated with longevity in humans. Five SNPs, four of which are haplotype-tagging, were selected from the public databases and genotyped in our extensive DNA collection of more than 1500 long-lived

Frequencies of the common five-SNP haplotypes in the SIRT1 gene

Haplotypea Frequencyb in controls Frequencyb in LLI P Frequencyb in P haplotype haplotype centenarians 11111 0.580 0.570 0.59 0.558 0.35 22121 0.255 0.261 0.74 0.275 0.34 11112 0.083 0.087 0.70 0.091 0.55 22221 0.064 0.062 0.83 0.062 0.86 11121 0.013 0.018 0.32 0.012 0.80 Global significance 0.58c 0.90c

a The five-SNP haplotype block as defined by Haploview. The SNPs in the haplotypes are listed in the 50 to 30 order; 1 denotes the major allele, 2 the minor The first and the second SNP are in perfect LD and provide the same haplotype information. SNPs 2 to 5 are haplotype-tagging. b Frequencies inferred by the EM algorithm using the program COCAPHASE. c Global significance value obtained after 1000 permutations with COCAPHASE.

gene were identified, but no association between these and exceptional longevity was observed. Neither could we detect association at the single-point allele or genotype levels.

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