association, indicating that DNA-damage response and repair overall appears to be important for human longevity. In the subsequent competitive gene-set analysis, we found nominal significant associa- tion with longevity of three sub-processes: BER, HRR and RECQ, which indicates that these may be of greater importance compared with the remaining six sub-processes. Notably, the lack of comparative significance for the remaining sub-processes does not necessarily imply that these are irrelevant for longevity, but rather that they appear less important than BER, HRR and RECQ. When correcting for the test of nine sub-processes, only HRR remained significant and only in setup II (Table 2). One reason for the lack of significance for the remaining associations could be reduced power due to an inadequate sample size (1089 oldest-old and 736 middle-aged), which implies that larger study populations may be needed for a more conclusive result. Then again, the lack of significance could well be an inherent part of the competitive analysis as we compare the associations of sub-processes all of which are considered relevant for longevity; thus, it may be difficult to uncover a significant relative difference between such sub-processes. Our findings indicate that repair of certain types of DNA damage likely could be of particular importance for human longevity; for instance damage induced by ROS or ionizing radiation, which is repaired by BER and HRR, respectively. Moreover, ROS induced damage can give rise to DNA DSBs during DNA replication, which potentially induce replication stress and replication fork collapse, a

An issue of increasing interest in genetic association studies is gene- environment (G(cid:2) E) interactions. It is, though, problematic to consider environmental factors in case-control studies of longevity; these factors may not be constant over time, that is, the longevity cases could have experienced a different environment when they were at the age of the younger controls. Instead, a longitudinal design that investigates survival during old age would be more suitable for G(cid:2) E studies. In relation to DNA repair, a G(cid:2)E analysis could be especially appealing as the sub-processes repair certain types of DNA damage induced by certain types of environmental sources. An example is the oxidative damage repaired by BER, which, among others, is induced by the general metabolism, which might be affected differently by diverse types and amounts of food intake.

difference between such sub-processes. Our findings indicate that repair of certain types of DNA damage likely could be of particular importance for human longevity; for instance damage induced by ROS or ionizing radiation, which is repaired by BER and HRR, respectively. Moreover, ROS induced damage can give rise to DNA DSBs during DNA replication, which potentially induce replication stress and replication fork collapse, a situation in which HRR is crucial. Finally, the roles of the RECQ helicases for ensuring the proper structure of the DNA during repair could be central. This is especially interesting considering the existence of premature aging syndromes, such as the Werner syndrome, caused by mutations in a RECQ gene.

When we performed the self-contained set-based analysis (of all SNPs under study) and the competitive gene-set analysis of BER, HRR and RECQ in the German study population, we did not observe replication. There are a number of possible reasons for this. First of all, different genotyping procedures were used in the two study populations. This may cause differences in the coverage of the variation in the gene regions, which potentially could affect the relative difference between the sub-processes. Specifically, five genes were not covered at all in the German data: H2AFX (DNA-damage response), APEX1 (BER), ACD (telomere functioning), ERCC1 (nucleotide excision repair) and RECQL4 (BER, RECQ, telomere functioning and mtDNA processes). Moreover, not exactly the same SNPs were genotyped in the two study populations; hence, depending on the LD of the discovery SNPs, these might not all be tagged in the German data. Secondly, we aimed to replicate very modest-sized enrichment score P-values (P¼0.004–0.048, Table 2), of which only one in nine remained significant after correcting for the test of nine sub-processes. Thus, it may be difficult to replicate associations with such values as compared to replicating those with smaller P-values. Furthermore, it might be difficult to detect a relative significance between sub-processes (competitive gene-set analysis) in the German data set considering that the overall pathway was non-significant (self- contained set-based analysis). Thirdly, the Germans were older than the Danes, and, if the associations are not constant throughout old age, they may not replicate well. Such change in association with age has been reported previously.52 Finally, it is possible that the effect of BER, HRR, RECQ and of the pathway overall is population specific, perhaps mediated through population-specific characteristics in environment. Such population-specific effects have previously been suggested for single variants.53 Hence, before final conclusion can be drawn, additional studies in other, preferably larger, study