3. Results

By conducting a thorough literature and data base search candi- date genes, candidate SNPs and tagging SNPs were identified (see Sections 2.2 and 2.3 for details). A total of 148 candidate genes and 1273 SNPs were successfully genotyped and are listed in Supplemen- tary Table 1: these constitute 592 SNPs in 77 genes of the DNA dam- age signaling and DNA repair pathway, 370 SNPs in 33 genes of the GH/IGF-1/INS signaling pathway and 311 SNPs in 38 pro/antioxidant genes. The characteristics of the Danish discovery cohorts of middle- aged and oldest-old individuals are described in Table 1A, while the German and Dutch replication samples are described in Table 1B.

Next the 6 SNPs were evaluated in a replication cohort of 563 85+ year old Dutch individuals. The frequency data of the 6 SNPs in the Dutch and Danish longitudinal cohorts are listed in Supple- mentary Table 6. Applying an additive model, none of the SNP associ- ations was replicated in the Dutch cohort (see Table 5). However, inspection of Kaplan–Meier survival plots for the 6 SNPs suggested a recessive mode of effect for rs10047589 (TNXRD1) in the Dutch data (data not shown), with a similar direction of effect as in the Danish

3.1. Case–control comparison of middle-aged and oldest-old Danes

Exploiting the case–control allele comparisons revealed consider- ably more nominally significant differences than would be expected by chance (see Supplementary Table 2). After adjusting allelic com- parisons for multiple testing by permutation, 10 of the 1273 SNPs remained significant; of these SNPs the minor allele of the GH/IGF-

Table 2

significantly associated with longevity after correction for multiple testing in the case–control comparison of middle-aged and oldest-old Danish cohorts. (A) Both genders and (B) additional SNP in sex-stratified analyses.

Gene SNP Minor/major Chr./position Position in MAF MADT/1905 PCCA PCCA OR (95% CI) allele gene uncorrected corrected (A) Both genders Pro/antioxidants GSR rs1002149 A/C 8/30705280 5′ 0.183/0.137 1.47·10−4 0.0335 0.706 (0.589–0.845) GH/IGF/INS INS rs3842755 A/C 11/2137372 3′ UTR 0.292/0.424 8.38·10−16 1·10−4 1.789 (1.552–2.063) KL rs1207362 A/C 13/32510839 Intronic 0.339/0.245 2.77·10−9 1·10−4 0.634 (0.545–0.737) GHRHR rs2267723 G/A 7/30973467 Intronic 0.414/0.330 2.28·10−7 1·10−4 0.696 (0.607–0.799) GHSR rs572169 A/G 3/173648421 Coding (syn) 0.342/0.274 1.65·10−5 0.0045 0.725 (0.626–0.839) IGF2R rs9456497 G/A 6/160363418 Intronic 0.191/0.252 1.86·10−5 0.0050 1.424 (1.211–1.674) DNA repair RAD52 rs11571461 G/A 12/896580 Intronic 0.098/0.195 5.05·10−15 1·10−4 2.226 (1.815–2.730) WRN rs13251813 A/G 8/31083753 Intronic 0.077/0.134 1.72·10−7 2·10−4 1.840 (1.460–2.318) POLB rs2953983 G/A 8/42332413 Intronic 0.080/0.123 4.54·10−5 0.0225 1.599 (1.274–2.006) RAD23B rs1805329 A/G 9/109124149 Coding (non-syn) 0.245/0.190 6.74·10−5 0.0309 0.722 (0.615–0.847) (B) Males DNA repair NTLH1 rs3211994 A/G 16/2029029 3′ 0.112/0.201 1.38·10−5 0.0056 1.994 (1.455–2.732)

Notes: Chr: chromosome, MAF: minor allele frequency, MADT: the Study of Middle Aged Danish Twins, 1905: the Danish 1905 Birth cohort, PCCA: allelic case–control comparison P-value, OR: odds ratio, 95% CI: 95% confidence interval, UTR: untranslated region, Syn: synonymous, Non-syn: Non-synonymous.

longitudinal study, although the association was not significant; HR=0.758, p=0.103, 95%, CI=[0.543–1.058].

variations and/or genes. The investigation of large collections of can- didate variations grouped together based on the functional roles of the gene products is a much more recent strategy (Flachsbart et al., 2010; Nebel et al., 2009; Pawlikowska et al., 2009b). In this study we investigated 592 SNPs in 77 genes of the DNA damage signaling and DNA repair pathway, 370 SNPs in 33 genes of the GH/IGF-1/INS signaling pathway and 311 SNPs in 38 pro/antioxidant genes, making it the most comprehensive study to date following this strategy. Fur- thermore, in addition to the widely used case–control approach, we also present data from a longitudinal analysis of survival in the oldest-old.

We wanted to explore whether the 11 SNPs identified via the Danish case–control study maintained an effect during old age. Conse- quently we investigated the 11 SNPs in the longitudinal data on the oldest-old Danes.Similardirections of effects,i.e. positive effects onsur- vival, were noted for 5 SNPs; when applying the number-of-days-lived variable, rs13251813 (WRN) and rs3842755 (INS) displayed nominally significant associations (pb0.05), the former in the entire group of oldest-old and the latter when restricting to males. Additionally, rs9456497 (IGF2R) showed a borderline significant association when restricting to males (p=0.05). For the early/late death variable, rs3211994 (NTHL1) displayed nominal significance in both genders combined, while rs1157146 (RAD52) showed a non-significant tenden- cy in females only (p=0.10). None of the remaining six SNPs showed similar directions of effect. The data is summarized in Table 6A.

In the analysis of the case–control study of 1089 oldest-old and 736 middle-aged Danes, we found 11 novel SNPs in 11 different genes to be associated with human longevity. The rare allele of vari- ants in INS, IGF2R, POLB, RAD52, WRN and NTHL1 was demonstrated to be advantageous, the latter most evident in males, whereas the rare allele of variants in KL, GHRHR, GHSR, GSR, and RAD23B was dis- advantageous for survival.