2.1.Differences between centenarians and controls were analyzed, but no evidence of association was found, even when gender-stratified samples were used. The majority of the genetic variation present within the LD block was captured by selecting five markers of varying minor allele frequency spread across the SIRT1 region. These markers were spaced across the coding region, the 3 UTR, and the promoter of the gene. The promoter of the SIRT1 gene was localized within the genomic sequence NM_012238 to between about 500 and 1000 bp downstream of hCV3003909. The five SNPs defined five haplotypes, with a frequency of over 1% in the control population, and with the three most frequent haplotypes accounting for 92% of the data. Four of these markers were haplotype-tagging SNPs (htSNPs), meaning that they were sufficient to resolve the five haplotypes. None of the five-SNP haplotypes differed significantly in frequency between case samples and controls. Single marker case-control analyses on allele and genotype frequency data were performed using contingency table chi-square 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/haploview/) was used to assess all polymorphisms for significant deviation from Hardy-Weinberg equilibrium, calculate LD (D) between each marker pair, and visualize haplotype structure. Haplotype frequencies were inferred using the expectation maximization (EM) algorithm and the sliding window option 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. The SIR2/Sirt1 gene has been shown to regulate lifespan in many model organisms, including yeast, C. elegans, and rodents. In mammals, expression of the SIRT1 protein is activated by various stress stimuli, leading to a breakdown of fat stores, induction of cell cycle arrest and DNA repair processes, or attenuation of apoptosis. Under adverse circumstances, SIRT1 appears to increase organismal longevity by tipping physiological responses away from cell death and towards cellular stress resistance. 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, and therefore possibly humans. The entire collection of 1026 German LLI and the subset 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 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. Frequencies of the common five-SNP haplotypes in the SIRT1 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.Nature, Kaeberlein, M., Mey, M., Guarente, L. "The SIR complex and SIR alone promote longevity in Saccharomyces cerevisiae by two different mechanisms." Genes Dev., Lin, S.J., Defossez, P.A., Guarente, L. "Requirement of NAD and SIR for lifespan extension by calorie restriction in Saccharomyces cerevisiae." Science, Luo, J., Nikolaev, A.Y., Imai, S., Chen, D., Su, F., Shiloh, A., Guarente, L., Gu, W. "Negative control of p by Sir alpha promotes cell survival under stress." Cell, Masoro, E.J. "Caloric restriction and aging: an update." Exp. Gerontol., Motta, M.C., Divecha, N., Lemieux, M., Kamel, C., Chen, D., Gu, W., Bultsma, Y., Murney, M., Guarente, L. "Mammalian SIRT represses forkhead transcription factors." Cell, Nebel, A., Croucher, P.J., Stiegeler, R., Nikolaus, S., Krawczak, M., Schreiber, S. "No association between microsomal triglyceride transfer protein (MTP) haplotype and longevity in humans." Proc. Natl. Acad. Sci. USA , Nemoto, S., Fergusson, M.M., Finkel T. "Nutrient availability regulates SIRT through a forkhead-dependent pathway." Science , Onyango P., Celic I., Ma ffery J.M., Boeke J.D., Feinberg A.P. "The htSNP approach employed here is likely to have captured the common variation present in the SIRT1 genomic region for the samples analyzed in this study. By tagging the common variation (haplotypes) within SIRT1, we implicitly assume that any undetected rare variants will be carried by one or other of the common background haplotypes and hence be statistically detectable; this is the logic of the htSNP approach. It is of course possible, albeit unlikely, that the selected markers might not tag the common variation comprehensively, and that rare SIRT1 polymorphisms with a weak impact on the longevity phenotype may exist. Overall, however, our findings would suggest that there is no noteworthy influence of SIRT1 gene variation on exceptional human longevity in the German population. The possibility that this negative association finding results from population stratification in our samples is unlikely. The validity and efficacy of our extensive study population for genetic longevity research have recently been demonstrated (Nebel et al., 2005)." The conserved function of SIRT1 in invertebrates and mammals predicts that the up-regulation of the SIRT1 gene and the subsequent increase in levels of the SIRT1 protein would also exert a life-prolonging effect in humans. In this case, potential mutations affecting the transcription of the gene would be of particular interest. Because the htSNPs used in this study, which are in strong LD, also span the promoter of SIRT1, a significant influence of variation in this