From a pathway perspective, a few comments can be made on the genes holding the SNPs suggested here. For the GH/IGF-1/INS pathway, the present data suggest that variations in genes whose products 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 baseAlternatively, 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 underpowered replication populations. However, 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 Danish 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. Changes in association effects with age have 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 the environment. Such population-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 for 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. Hence, we did not correct for performing sex-stratified analysis as well as a test for both genders combined and with respect to the longitudinal study for investigating the two survival variables. Such corrections should, in principle, be included to avoid false positive findings.

If correcting the p-values from each pathway using the overly conservative Bonferroni approach, 8 of the 11 SNPs from the case-control study remained significant, while 1 of the 6 SNPs identified in the longitudinal study remained significant. By contrast, correction by the less conservative Benjamini and Hochberg (1995) method left more associations significant (10 SNPs from the case-control study and 1 SNP from the longitudinal study), thus underscoring the difference in conclusions based on the choice of method of correction. In any case, both of these correction methods assume independence between the tests, which is clearly not the case in our study.

Due to the emergence of high throughput techniques facilitating studies of sizable numbers of variations, the replication of initial findings has become a major issue in genetic epidemiology. In this study, we tested our findings from the case-control and longitudinal studies using German samples of 1613 long-lived individuals (95+) and 1104 middle-aged controls and a cohort of 85+ Dutch elderly, respectively. 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 an SNP initially identified in the Danish longitudinal study.

(A) Case-control study (initial findings) Longitudinal study (similar directions of effects) 
Number-of-days-lived variable Pathway Gene SNP OR -coef. 95% CI P-value 
GH/IGF-1/INS INS rs3842755 1.789 2.73 0.66-4.80 0.01 
IGF2R rs9456497 1.424 2.77 0.015-5.56 0.05 
DNA repair WRN rs13251813 1.840 2.23 0.31-4.15 0.02 
Early/late death variable Pathway Gene SNP OR OR 95% CI P-value 
DNA repair RAD52 rs11571461 2.226 1.30 0.96-1.78 0.10 
NTHL1 rs3211994 1.994 1.42 1.09-1.86 0.009 

(B) Longitudinal study (initial findings) Case-control study (similar direction of effect) 
Number-of-days-lived variable Pathway Gene SNP -coef. OR 95% CI 
PCCA Pro/antioxidants XDH rs207444 4.043 1.42 1.10-1.83 0.005 

Notes: OR: odds ratio, -coef: beta coefficient, 96% CI: 95% confidence interval, : males separately, : females separately, PCCA: allelic case-control comparison P-value.

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