Interestingly, the longitudinal study revealed more longevity- associated SNPs in the sex-stratified analysis (4 of 6 SNPs) than the Danish case–control study (1 of 11 SNPs). This observation could in- dicate that the impact of genetic effect becomes more gender- specific during advanced age than at younger ages, and that the ge- netic effect in old age might be most pronounced for males. Such a difference between genders was recently suggested by Montesanto et al. (2011) for Italians.

From a pathway perspective a few comments can be made on the genes holding the SNPs suggested here. For the GH/IGF-1/INS path- way the present data suggests that variations in genes whose prod- ucts take part in growth hormone synthesis and secretion (GHRHR, GHRL and GHSR) or in signaling to and affecting target organs/cells (INS, KL, IGF2R) might pose effects on longevity. With respect to DNA repair, the gene products of the genes holding the significant SNPs take part in different DNA repair activities, though the majority take part in base excision repair (NTHL1, POLB, and WRN) or in DNA double strand break repair (RAD52, H2AFX, XRCC5 and WRN), thus in- dicating that variation in the repair of DNA damage induced by for in- stance reactive oxygen species and ionizing radiation could pose effects on longevity. Lastly, with respect to the pro/antioxidant path- way both antioxidants (GSR and TXNRD1) and a prooxidant (XDH) are observed, indicating that the role of this pathway in longevity appears complex.

Table 5

Replication of findings from the Danish longitudinal study in the longitudinal Dutch Leiden 85-plus Study.

Both genders combined Danish longitudinal study Dutch longitudinal study Pathway Gene SNP Estimate HR P-value 95%CI Pro/antioxidants TNXRD1 rs10047589 1.458 (OR) 0.969 0.659 0.844–1.112 Pro/antioxidants XDH rs207444 4.043 (β) 0.886 0.404 0.667–1.177 Males GH/IGF-1/INS GHRL rs26802 2.338 (OR) 0.730 0.962 0.774–1.197 DNA repair MLH1 rs13320360 3.132 (OR) 1.169 0.827 0.288–4.727 XRCC5 rs705649 4.669 (β) 1.037 0.749 0.828–1.300 Females DNA repair H2AFX rs2509049 0.625 (OR) 0.909 0.260 0.769–1.073

Notes: β: Beta coefficient (linear regression using the number-of-days-lived variable), OR: odds ratio (logistic regression using the early/late death variable), HR: hazard ratio, 95%CI: 95% confidence interval.

TheSNPssuggested hereto associate to longevitymightinduce dif- ferent functional effects; rs1002149 (GSR) and rs2509049 (H2AFX) lo- cated 5′ of genes might affect the level of gene expression, while rs3842755 (INS), rs3211994 (NTHL1) and rs10047589 (TNXRD1) are all located 3′ and could thus in principle influence decay of mRNA. The non-synonymous coding SNP rs1805329 (RAD23B) could poten- tially affect protein activity, while the intronic SNPs (rs1207362 (KL), rs2267723 (GHRHR), rs9456497 (IGF2R), rs11571461 (RAD52), rs13251813 (WRN), rs2953983 (POLB), rs207444 (XDH), rs26802 (GHRL), rs13320360 (MLH1) and rs705649 (XRCC5)) could possibly induce splicing alterations. Alternatively, the associated SNPs might simply be proxies for other truly causative variants.

A number of reasons can be given for the lack of true replication. It could be a matter of under-powered replication populations. Howev- er, the number of German samples is larger than the Danish discovery cohorts, thus arguing against this explanation. Alternatively it might be due to heterogeneity between samples, for instance differences in enrollment of the participants. As seen in Table 1, the German cases are older than the Danish oldest-old, whereas the members of the Dutch 85+ cohort are somewhat younger at intake than the Dan- ish 1905 cohort members. Hence, if the effects of the variants are not constant throughout old age, they may not replicate well in these study samples. Change in association effects with age has been reported previously (Jacobsen et al., 2010). Finally, it might simply be that the associations are population specific, perhaps mediated through population specific characteristics in environment. Such pop- ulation specific effects have previously been suggested (Di Bona et al., 2009).

How such suggested functional effects might impact aging and/or longevity is an extremely difficult question, especially considering that the role of the three pathways in aging and/or longevity appears very complex. In any case, functional studies of the molecular effects of the associated variants are needed

In our study we only performed correction of multiple testing within each pathway, since the selection of genes and SNPs was based on a sound a priori hypothesis. Moreover, due to the design of the statistical software, we corrected for multiple testing within each type of test, and hence, we did not correct for performing the sex-stratified analysis as well as a test for both genders combined, and with respect to the longitudinal study for investigating the 2 sur- vival variables. Such corrections should in principle be included, in order to avoid false positive findings. If correcting the p-values from

Due to the emergence of high throughput techniques facilitating studies of sizeable numbers of variations, the replication of initial find- ings has become a major issue in genetic epidemiology. In this study we tested our findings from the case–control and the longitudinal study using German samples of 1613 long-lived individuals (95+) and 1104middle-aged controls,and a cohortof 85+ Dutchelderly,re- spectively. No formal replication was observed in either case although a few supportive observations were found (see above).

Table 6

Similar directions of effect in the Danish case–control and longitudinal studies.

(A) Similar directions of effect in the Danish longitudinal study of SNPs initially identified in the Danish case–control study and (B) Similar direction of effect in the Danish case– control study of a SNP initially identified in the Danish longitudinal study.