None of the findings could be formally confirmed in a German rep- lication cohort of comparable size, although rs11571461 (RAD52) did show a tendency in the same direction as found in the Danish data. In support of our findings, other variants in KL, WRN and NTHL1 have previously been associated with longevity (Arking et al., 2005; Castro et al., 2000; Yashin et al., 2010) and rs687045 (KL) which was put forward by Lunetta et al. as a mortality SNP during old age (Lunetta et al., 2007), is in our cohorts in moderate LD (R2=0.25) with rs1207362 (KL) described in our study as disadvantageous for longevity. Moreover, supportive of an association of INS, a very recent publication of gene-based case–control analysis of genes in the IGF-1/ Insulin pathway reported gene-based association of INS with longev- ity (Deelen et al., 2011). Additionally, some of the 11 SNPs have been

Finally, in order to explore whether the 6 SNPs, identified in the longitudinal study of oldest-old Danes, presented a similar direction of effects on survival from middle age to old age, we investigated the 6 SNPs in the Danish case–control data. Only rs207444 (XDH) dis- played a nominally significant effect in the same direction as found in the oldest old (p=0.005). The data is summarized in Table 6B.

4. Discussion

The vast majority of the candidate genetic association studies longevity published so far has explored only a small number of

Table 3

Replication of Danish case–control findings in the German case–control samples.

Gene SNP OR (Danish study) OR (German study) PCCA Germans 95% CI Pro/antioxidants GSR rs1002149 0.706 1.054 0.470 0.913–1.217 GH/IGF/INS INS rs3842755 1.789 0.949 0.434 0.832–1.082 KL rs1207362 0.634 0.985 0.804 0.873–1.110 GHRHR rs2267723 0.696 1.005 0.934 0.900–1.121 GHSR rs572169 0.725 1.106 0.097 0.981–1.247 IGF2R rs9456497 1.424 0.962 0.586 0.837–1.105 DNA repair RAD52 rs11571461 2.226 1.162 0.191 0.927–1.457 WRN rs13251813 1.84 0.999 0.998 0.754–1.325 POLB rs2953983 1.599 1.046 0.683 0.842–1.299 RAD23B rs1805329 0.722 0.955 0.507 0.833–1.094 DNA repair (males) NTHL1 rs3211994 1.994 0.931 0.627 0.699–1.241

Notes: OR: odds ratio, PCCA: allelic case–control comparison P-value, 95% CI: 95% confidence interval.



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reported as associated with phenotypes of possible relevance to vari- ation in lifespan; rs572169 (GHSR) with obesity (Gueorguiev et al., 2009), and rs2953983 (POLB), rs1805329 (RAD23), rs11571461 (RAD52) and rs13251813 (WRN) with different types of cancers (Pan et al., 2009; Schildkraut et al., 2010; Van Ness et al., 2008; Yang et al., 2009).

An interesting question was whether the genetic variants with a proposed effect on survival from middle age to old age would also demonstrate an effect for survival during old age. We therefore tested the 11 SNPs for association to survival in the Danish 1905 cohort using the longitudinal design and found 3 SNPs to display the same nominal significant (pb0.05) direction of effect in the longitudinal data, while 2 SNPs showed similar tendencies (0.05bpb0.10). Inter- estingly, all 5 SNPs were apparent longevity SNPs, i.e. SNPs with a positive effect on survival. This is in line with a recent study suggest- ing that long life is not due to a decreased burden of negatively affect- ing disease variants but rather due to positively influencing longevity variants (Beekman et al., 2010).

A lack of similar effects in the longitudinal data for the remaining SNPs could indicate that these SNPs may be relevant at different ages, i.e. the influence of genes and gene variants may be of consequence for survival in young elderly, but lose this importance at the highest ages.

When investigating the association of the 1273 SNPs with survival at advanced age using a longitudinal study approach, we identified 6 novel SNPs in 6 genes to be associated with longevity. The rare allele of a variant in H2AFX appeared disadvantageous, while the rare vari- ants in GHRL, MLH1, TXNRD1, XDH and XRCC5 were advantageous for longevity. In line with these results an MLH1 haplotype was previous- ly reported to be associated with longevity in a case–control study (Kim et al., 2006), whereas genetic variants in GHRL have been de- scribed as associated with phenotypes of possible relevance to varia- tion in lifespan; high HDL-cholesterol levels (Hubacek et al., 2007) and hypertension (Berthold et al., 2010). Finally, the activity of the XRCC5 protein product Ku80 has been reported to decrease from young to old age in humans (Scarpaci et al., 2003), indicating a bio- logical change with age.

The Danish case–control study supported an advantageous effect of rs207444 (XDH) also on survival from middle age to old age, and additionally some support was observed for rs10047589 (TNXRD1) in the Dutch replication cohort, indicating a recessive mode of effect for this variant.