First, the influence of environmental factors could not be controlled in this study due to lack of information in the control group. Second, we could not evaluate the function of any SNP that was not a missense mutation but did have statistical evidence for association. Third, given the small number of centenarians, the statistical power may not be sufficient to significantly detect genes with weak effect, particularly, for the gene-gene interac- tion. There are further issues concerning the inter- pretation of the findings using the over-dominant model and the control group consisted of 12-14 yr old middle school students. To eliminate the impact of different substructure between the long-lived group and the young control group, the P values were corrected by genomic control for each age-gender group. In the nonagenarian group, the Bonferroni threshold for P ＜0.05 with 463 SNPs was 1.08 ×10-4 and the significance level was relaxed by 1000 permutations to 1.69 × 10-4 , 1.56 ×10-4 , 2.16 ×10-4 , 1.50 ×10-4 , and 2.42 ×10-4 under the co-dominant, dominant, recessive, over-dominant, and log-additive models, respec- tively. None of the SNPs achieved genome-wide significance. However, the underlying LD structure among the markers reduced the number of effec- tively independent tests, and so the genome-wide threshold of significance may be too stringent particularly in the context of candidate gene approach (Rao and Gu, 2001).

According to the theory of evolution, differential survival reflects the effect of deleterious genotypes of age-related diseases. However, some longevity genes (e.g. klotho that is associated with low HDL) buffer the deleterious effect of age-related diseases genes (e.g. lipoprotein, Lp(a), a susceptibility gene for vascular disease), which may explain an observed paradoxical increase of deleterious geno- types in centenarians (Bergman et al., 2007). Lunetta et al. (2007) conducted a genome-wide association study of age-related phenotypes using Affymetrix 100 K SNP Chips in the context of a cross-sectional study design. However, none of the associations with 5 aging traits achieved geno- me-wide significance using 1,345 Framingham Study

In the current study, a total of 60 genes out of 179 candidates were nominally associated (P ＜ 0.05) with longevity in at least one of age/gender groups. Among eleven genes that consistently yielded statistical evidence of allelic, genotypic and haplotypic association with lifespan, only four genes remained significant after the Bonferroni correction for haplotypes. The protein encoded by

drew a 10 ml fasting venous blood sample in an EDTA tube. The institutional review board (IRB) of the Samsung medical center reviewed the research protocols and approved this study. The gender and age composition of study subjects were shown in Supplemental Data Table S1.

For genetic variants with strong effects, relati- vely small sample size is sufficient to achieve statistical power to detect association. The results in this study warrant further replication studies. However, the expectation for replication of results may need to be relaxed in studies of extremely old subjects, who are rarely observed. Under such circumstances, additional information gathered from laboratory techniques, bioinformatics, and a priori insights of biological pathways could be used to provide plausibility for interpreting genetic associa- tion findings (Chanock et al., 2007). To increase statistical power of a test for gene by gene or gene by environment interaction, international collabora- tion is essential due to the rarity of centenarians. Continuing efforts may be necessary to find a functional variant in the associated region.

Candidate genes and SNP selection

Candidate genes, mostly for DM, CVD, and cancers, were selected based on searches for their functions in public databases (OMIM, Gene, etc.) and previous literature reviews. SNP markers within or near candidate genes were identified from the literature, the dbSNP database (http://www.ncbi.nlm.nih.gov/projects/SNP/), the HapMap Ⓡ database (http://www.hapmap.org/), and Vector NTI soft- ware (Invitrogen, Inc., Carlsbad, CA). We included 565 informative single nucleotide polymorphism (SNP) markers with high design scores provided by Illumina, Inc. (San Diego, CA) and heterozygosity above 0.05 using 300 independent healthy Korean samples. Information for these 565 SNPs located in 194 genes is presented in Supplemental Data Table S2.

Nevertheless, the results from various statistical analyses consistently supported that the presence of the target allele of a potent SNP conferred a greater likelihood of longevity in Koreans. The effects of gender mediated genetic association with the probability of achieving longevity, as shown in previous studies (Franceschi et al., 2000) and the observed gender difference in our findings supports, in part, the gender-specific probability of achieving longevity. Interestingly, a few candidate gene va- riants with no or insignificant allelic effect revealed strong evidence for the allele with a large hetero- zygote effect on longevity (e.g. MGP, TNFSF11, and ABCC8).

DNA preparation and SNP genotyping

Genomic DNA samples were prepared from peripheral blood using the QIAamp DNA Blood Maxi kit following the manufacturer's instructions (Qiagen, Inc., Valencia, CA). For each sample, an average concentration of DNA from three repeated measures was determined using a Spec- traMax Plus 384 spectrophotometer (Molecular Devices, Inc., Sunnyvale, CA). A 250 ng aliquot of DNA was genotyped for SNP markers using Golden-Gate chemistry on SentrixⓇ Array Matrices (Illumina, Inc.) at the Samsung Biomedical Research Institute. Genotypes showing signi- ficant statistical evidence for association with longevity were confirmed using an ABI PRISM 3100 sequencer (Applied Biosystems, Inc., Foster City, CA).