The frequency of centenarians is approximately 1/100,000 people worldwide. According to the Korea National Statistical Office (KNSO) Report in 2005 (http://www.kosis.kr.), the frequencies of nona- genarians and centenarians were 131 and 2/100,000 Koreans, respectively (KNSO, 2005). Centenarians may not be free of disease, but manage to survive with or without treatment owing to their resistance to disease (Franceschi et al., 2000). The incidences of major common diseases slow down or even decline around age 85-90 yr (Bergman. et al., 2007). Gender also accounts for important differences in longevity as well as in the occurrence of a variety of age-related disease. The male/female ratios were about 1:4 in nonagenarians and 1:8.2 in centena- rians in Korea (KNSO, 2005). Such data raise the possibility of gender-specific genetic determinants of human longevity.

Because the difficulty in finding centenarians or nonagenarians limits the replication of genome-wide association (GWA) studies using an extremely large number of randomly spaced markers, we adopted a high-throughput candidate gene approach to identify susceptibility variants controlling lifespan in Koreans.

Results

Approximately 30 candidate genes for longevity have been reported in previous studies since Herman (1956) proposed the free radical theory of aging, where oxidative stress causes aging (Salvioli et al., 2006). Genes involved in inflamma- tion and the immune response (e.g. IL6; LMP2, low molecular mass protein 2; TNFA, tumor necrosis factor (TNF superfamily, member 2); TGFB1, trans- forming growth factor, beta 1; and PPARγ, pero- xisome proliferator-activated receptor gamma), genome maintenance and repair (e.g. P53), lipid metabolism (e.g. APOE, apolipoprotein E; CETP, cholesteryl ester transfer protein, plasma; MTTP, microsomal triglyceride transfer protein, and PON1, paraoxonase 1), glucose metabolism (e.g. IGF1, insulin-like growth factor 1 (somatomedin C); and HFE, hemochromatosis), oxidative stress (e.g. SOD1, superoxide dismutase 1, soluble; PARP, poly (ADP-ribose) polymerase 1; and GSTT1, gluta- thione S-transferase theta 1), mitochondrial muta- tion, premature aging syndromes such as Werner

(18%) with MAF ＜ 2%, HWE P ＜ 10-4 , or CR ＜ 95% in the young age group did not passed the threshold of our quality test for genotypes. Thus, a total of 463 informative markers located in 176 genes were included in the subsequent analyses. For the comparison of the individuals above age 90 versus the young healthy controls, our study had a sample size adequate to reach a power of 80% to significantly detect a genotypic OR over 1.7/2.4 (heterozygote/ homozygote) under the assumptions shown above (see the Methods). The result obtained from centenarian women was shown in Supplemental Data Tables.

Allelic- and genotypic association

A total of 43 SNPs located in 33 genes (18.8% of 176 candidate genes) had a nominally significant association with lifespan (P ＜ 0.05) in the allelic χ2

tests (data not shown). Among these, 34 SNPs of 28 genes showed a significant allelic OR and 7 genes had a P value less than 0.01 in at least one age/gender group (i.e. IL12RB2, interleukin 12 receptor, beta 2; PPP1R14C, protein phosphatase 1, regulatory (inhibitor) subunit 14C; EGFR, epider- mal growth factor receptor; PAX4, paired box 4; MMP1, matrix metallopeptidase 1 (interstitial colla- genase); PPP1R1A, protein phosphatase 1, regu- latory (inhibitor) subunit 1A; and ALDH2, aldehyde dehydrogenase 2 family (mitochondrial)). A total of 128 SNPs located in 72 genes (41% of 176 genes) were statistically associated with one or more age/gender groups (P ＜ 0.05) using regression models for five modes of inheritance (data not shown). Among these, the genotypic ORs of 95 SNPs located in 68 genes were nominally signi- ficant in at least one of age/gender groups and 13 genes had a P value less than 0.01 (Table 1). We present the P values corrected by genomic control where the λ is greater than 1 (i.e. female cente- narians, log-additive model, λ = 1.05). As shown in Table 1, the genotypic effect on longevity was stronger than the corresponding allelic effect in either direction (i.e. either increased or decreased

ryotic translation initiation factor 4 gamma, 1 (EIF4G1), ATP-binding cassette, sub-family C (CFTR/MRP), member 8 (ABCC8), ALDH2, and telomerase-associated protein 1 (TEP1) showed evidence for genotypic association and 9 genes yielded P values less than 0.01 (Table 1B). By way of example, a male with an A allele of the rs671 (Lys504Glue) located in the ALDH2 increased the chance of living longer than 90 yr by 2.11 times compared to men having a G allele (P = 0.009) and this SNP fitted best under a dominant model (OR 2.63, P = 0.004). No allelic effect of the rs757110 (Ser1369Ala) in the ABCC8 gene was identified; however, the effect of the TG heterozygous geno- type was greater than that of the rare GG homo- zygous type (OR 3.12, P = 6×10-4 ).

Nonagenarian women

A total of 37 SNPs located in 31 genes showed evidence for genotypic associations in either age group of women (i.e. women aged over 90 yr and centenarian women). Genes such as PPP1R14C (P = 0.005) and the EGFR (P = 0.007) had lower allelic P values in the centenarian group with a small sample size (data not shown), while IL12RB2 was significant only in the younger group with more subjects (P = 0.008). However, the rare T allele of rs2030071 was not sufficiently common to deter- mine the genetic model (Table 1C).