As far as we know, this is the first association study of longevity susceptibility genes adopting a high-throughput candidate gene approach in nonagenarians and centenarians who rarely appear in Korea. An understanding of the mechanisms underlying aging and longevity by discovering novel susceptibility genes may lay the foundations for prediction, prevention, and treatment of age-related diseases. Thus, the findings of the current study may provide a starting point to unravel genetic factors controlling longevity for future studies.

Statistical analysis:

At each SNP, we separately computed the genotyping call rate (CR ≥ 95%), minor allele frequency (MAF ≥ 2%), and deviation from Hardy-Weinberg equilibrium (HWE P ≤ 0.0001) in the long-lived group and the young healthy group. We determined ancestral alleles for each SNP using the dbSNP database. We used the SNPs-based whole genome association studies (SNPassoc) program implemented in the free downloadable statistical software environment R v. 2.6.2 (http://www.r-project.org) to perform quality tests for genotypes (Gonzalez et al., 2007). We performed preliminary analyses of linkage disequilibrium (LD) patterns using both D' and r2 and then selected a set of tagging SNPs within a gene for haplotype-based analysis using Haploview v. 4.0 (http://www.broad.mit.edu/mpg/haploview/index.php/).

Subjects:

We identified 137 long-lived individuals over 90 years old, including 35 centenarians, from the Korean Centenarian Study, which began in 1999 (Choi et al., 2003). We also selected 213 healthy middle school students from individuals who visited the Samsung Seoul Hospital for psychiatric tests in 2003 to be used for comparison. We included eight control samples from a three-generation family to test genotype quality. After obtaining informed consent from all subjects, a clinician interviewed study subjects.

We performed subsequent statistical analyses for each of four groups stratified by age and gender (nonagenarians, male nonagenarians, female nonagenarians, and female centenarians). We assessed allelic association with the chi-squared tests for individual SNPs and calculated the allelic odds ratio (OR) and 95% confidence interval (CI) in both adjusted and stratified analyses using publicly available subroutines 'genassoc' and 'gamenu' in the STATA/SE v. 9 software package (http://www.gene.cimr.cam.ac.uk/clayton/software/stata/).

We performed genotypic association tests and logistic regression analyses implemented in the SNPassoc package in five different genetic models: codominant, dominant, recessive, over-dominant, and log-additive. To account for the impact of substructure or genetic relatedness among the subjects, we estimated the 'inflation factor ()' for each age-gender group and corrected P values by genomic control (Devlin and Roeder, 1999).

We used general linear models for the regression of longevity on ambiguous haplotypes. Rare haplotypes with an allele frequency of less than 1% were combined in a group to be analyzed. We applied logistic regression models (LRMs) to perform two-way interaction analyses between two SNPs for each gene (Briollais et al., 2007). For each of four age/gender groups, we screened the most promising pairs of SNPs that yielded P ≤ 0.01 for the interaction likelihood ratio test (i.e. LRT = -Of the pair of SNPs, Chanock SJ, Manolio T, Boehnke M, Boerwinkle E, Hunter DJ, Thomas G., Hirschhorn JN, Abecasis G., Altshuler D, Bailey-Wilson JE, Brooks LD, Cardon LR, Daly M, Donnelly P, Fraumeni JF Jr, Freimer NB, Gerhard DS, Gunter C, Guttmacher AE, Guyer MS, Harris EL, Hoh J, Hoover R, Kong CA, Merikangas KR, Morton CC, Palmer LJ, Phimister EG., Rice JP, Roberts J, Rotimi C, Tucker MA, Vogan KJ, Wacholder S, Wijsman EM and Winn DM Collins FS replicated genotype-phenotype associations. Nature 2007;447:655-60. Choi YH, Kim JH, Kim DK (twice), Kim JW (twice), Lee MS (twice), Kim CH and Park SC reported distributions of ACE and APOE polymorphisms and their relations with dementia status in Korean centenarians. J Gerontol A Biol Sci Med Sci 2003;58:227-31. We computed the statistical power of our sample sizes for each genotype using the web browser 'Genetic Power Calculator' (http://pngu.mgh.harvard.edu/~purcell/gpc/cc2.html) under an additive model with the assumption of disease allele frequencies of 0.3 and D' of 1. We also assumed a longevity prevalence of 0.13% and 0.002% for the Korean nonagenarians and centenarians respectively (Purcell et al., 2003). Christensen K., Johnson TE and Vaupel JW discussed the quest for genetic determinants of human longevity: challenges and insights in Nat Rev Genet 2006;7:436-48. A, Berardelli M., Motta M., Monti D., Bonafe M., Ferrucci L., Deiana L., Pes GM, Carru C, Desole MS, Barbi C, Sartoni G., Gemelli C, Lescai F., Olivieri F., Marchegiani F., Cardelli M., Cavallone L., Gueresi P, Cossarizza A, Troiano L, Pini G, Sansoni P, Passeri G, Lisa R, Spazzafumo L, Amadio L, Giunta S, Stecconi R, Morresi R, Viticchi C, Mattace R and De Benedictis G. studied if men and women follow different trajectories to reach extreme longevity in the Italian Multicenter Study on Centenarians (IMUSCE). Aging (Milano) 2000;12: Supplemental data. The supplemental data include three tables and can be found with this article online at http://e-emm.or.kr/article/article_files/SP-41-11-01.pdf.

We appreciated the technical support of our staff En Jin Han, Hanee Kwon and Yonghun Lim at the Center for Genome Research of the Samsung Biomedical Research Institute. We also gratefully acknowledge the hard work of those involved in collecting samples. This work was supported by the Research Program of dual regulation mechanisms of aging and cancer from the Korea Science and Engineering Foundation (M1075604000107N5604-00110), the Korea Research Foundation for Health Science and Hallym University Research Fund 2009 (HRF-2009-000).