Computational analysis showed that the third amplicon of ATM promoter region contains two Sac II restriction enzyme sites; one of them is in the SNP (C/T) only when the T > C institution at nucleotide 116 is present (Fig. 2). Therefore, we developed PCR-restriction fragment length polymorphism (RFLP) to determine genotypes. Before genotyping was carried out, we examined the RFLP reliability, and both direct sequencing and Sac II restriction digestion detected 41 DNA samples. The concurrence rate of both methods was 100%, indicating that the PCR-RFLP method is reliable (data not shown).

(Supplement Tables 1 and 2), and a far higher frequency in LLIs was observed (Supplement Table 3). In addition, association analysis did not show any gender specificity (data not shown). The distribution of the observed genotypes in long-lived individuals and control subjects conformed to the Hardy-Weinberg equilibrium. Genotyping was conducted by PCR-RFLP in 789 long-lived individuals and 886 control subjects. Comparison of genotypic frequencies revealed that there is a significant association between the genotypes and longevity (P = 0.01), but not alleles (Table 2). LLIs obviously showed a higher frequency of CT genotype than controls (53.0% versus 45.9%, P = 0.004). Furthermore, the percentage of CT heterozygote increases with age in both LLIs and controls.

3.3. AP-2a binds to the SNP site to repress ATM transcription To examine whether the SNP regulates the ATM mRNA expression in vivo, we measured the ATM mRNA levels in peripheral mononuclear cells from human individuals by RT-PCR (McKinnon, 2004). The results from 42 healthy old individuals showed that TT genotype has the highest level of ATM mRNA expression than the other two genotypes (Fig. 3A), although no significant differences were found between CC and CT genotypes (CC versus TT, P = 0.001; CT versus TT, P = 0.006; CC versus CT, P = 0.818), but an obvious tendency in the level of ATM expression from CC to TT genotypes was observed (Supplement Fig. 1). These results indicate that the SNP regulates the ATM mRNA expression in human cells in vivo.

To figure out the mechanism in which the SNP regulates the ATM mRNA expression, we analyzed the sequences around the SNP and found that the SNP forms an activator protein 2a (AP-2a) binding site. Then, we examined the binding ability of AP-2a on the sequences of the SNP by electrophoretic mobility shift assay (EMSA). EMSA was carried out using purified human recombinant AP-2a protein and biotin-labeled probes with either DNA fragments of the C or T allele. AP-2a strongly bound to the oligonucleotide containing the C allele and weakly associated with the oligonucleotide probe with the T allele. Competition assays with unlabeled probe further confirmed a specific interaction between AP-2a and the C allele (Challenges and Insights. Nature Reviews Genetics, Giacconi, R., Cipriano, C., Albanese, F., Boccoli, G., Saba, V., Olivieri, F., Franceschi, C., Mocchegiani, E., The G/C polymorphism L-is useful to screen old subjects at risk for atherosclerosis or to reach successful aging. Experimental Gerontology, Herbig, U., Ferreira, M., Condel, L., Carey, D., Sedivy, J.M., Cellular senescence in aging primates. Science, Ito, K., Hirao, A., Arai, F., Matsuoka, S., Takubo, K., Hamaguchi, I., Nomiyama, K., Hosokawa, K., Sakurada, K., Nakagata, N., et al., Regulation of oxidative stress by ATM is required for self-renewal of hematopoietic stem cells. Nature, Leask, A., Byrne, C., Fuchs, E., Transcription factor AP and its role in epidermal-specific gene expression. Proceedings of the National Academy of Sciences of the United States of America, Liu, W.G., Smith, D.I., Rechtzigel, K.J., Thibodeau, S.N., James, C.D. Denaturing high-performance liquid chromatography (DHPLC) used in the detection of germline and somatic mutations. Nucleic Acids Research , Lombard D.B., Chua K.F. Mostoslavsky R. Franco S. Gostissa M. Alt F.W DNA repair genome stability and aging. Cell , Minnon P.J. ATM and ataxia telangiectasia second in molecular medicine review series. EMBO Reports , Puca A.A. Daly M.J. Brewster S.J. Matise T.C. Barrett J. Shea-Drinkwater M. Kang S. Joyce E. Nicoli J. Benson E., et al., A genome-wide scan for linkage to human exceptional longevity identifies a locus on chromosome Proceedings of the National Academy of Sciences of the United States of America, Shiloh, Y., ATM and related protein kinases: safeguarding genome integrity. Nature Reviews Cancer, Sun, T., Gao, Y., Tan, W., Ma, S., Shi, Y., Yao, J., Guo, Y., Yang, M., Zhang, X., Zhang, Q., et al., A six-nucleotide insertion-deletion polymorphism in the CASP promoter is associated with susceptibility to multiple cancers. Nature Genetics , Tyner S.D., Venkatachalam S., Choi J., Jones S., Ghebranious N., Igelmann H., Lu X.B. Soron G. Cooper B. Brayton C. et al., p mutant mice that display early aging-associated phenotypes. Nature , Wolford J.K., Blunt D., Ballecer C., Prochazka M., High-throughput SNP detection by using DNA pooling and denaturing high-performance liquid chromatography (DHPLC). Human Genetics , Christensen, K., Johnson T.E., Vaupel J.W. (2006). The quest for genetic determinants of human longevity: challenges and insights. Nature Reviews Genetics 7, 436.