Overall, the study investigates the association of common genetic variation in 148 genes related to GH/IGF-1/INS signaling, DNA damage signaling and repair, and pro/antioxidant pathways with human longevity. The study uses both case-control and longitudinal designs to investigate these variations in key candidate genes of human longevity. Replication studies were conducted in German and Dutch populations to validate the findings from the Danish study. The genotyping was performed using the Illumina GoldenGate platform with data cleaning according to manufacturer's recommendations. A total of 1273 SNPs were successfully genotyped and included in data analysis after exclusion criteria were applied.and the GH/IGF-1/INS-SNP rs3842755 (INS) were found to be associated with increased survival in females (see Table 4B). These results suggest that the genetic factors influencing survival in the oldest-old Danes may differ between genders. However, further replication studies in independent cohorts are needed to confirm these findings.survival. Our study is unique in that we investigated a large number of candidate genes and SNPs using both casecontrol and longitudinal approaches. Our analysis revealed that variants in genes involved in DNA damage signaling and repair, GH/IGF-1/INS signaling, and pro/antioxidant pathways are associated with human longevity. Specifically, the rare allele of variants in INS, IGF2R, POLB, RAD52, WRN and NTHL1 was advantageous for survival, while the rare allele of variants in KL, GHRHR, GHSR, GSR and RAD23B was disadvantageous. We also found sex-specific effects for some SNPs. However, our findings could not be formally confirmed in a German replication cohort of comparable size. Nonetheless, our study adds to the growing body of evidence suggesting that genetic variations play a significant role in human longevity.Longevity. Moreover, a recent publication of gene-based case-control analysis of genes in the IGF-1/Insulin pathway reported gene-based association of INS with longevity (Deelen et al., 2011). Additionally, some of the 11 SNPs have been reported as associated with phenotypes of possible relevance to variation 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).

Finally, 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) displayed a nominally significant effect in the same direction as found in the oldest old (p=0.005). The data are summarized in Table 6B.

4. Discussion

The vast majority of the candidate genetic association studies on longevity published so far has explored only a small number of SNPs. 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 tested the 11 SNPs for association with survival in the Danish 1905 cohort using the longitudinal design and found 3 SNPs to display the same nominal significant (p<0.05) direction of effect in the longitudinal data, while 2 SNPs showed similar tendencies (0.05<p<0.10). Interestingly, all 5 SNPs were apparent longevity SNPs, i.e. SNPs with a positive effect on survival. This is in line with a recent study suggesting that long life is not due to a decreased burden of negatively affecting 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 associated with longevity. The rare allele of a variant in H2AFX appeared disadvantageous, while the rare variants in GHRL, MLH1, TXNRD1, XDH and XRCC5 were advantageous for longevity. In line with these results, an MLH1 haplotype was previously reported to be associated with longevity in a case-control study (Kim et al., 2006), whereas genetic variants in GHRL have been described as associated with phenotypes of possible relevance to variation 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 biological 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.

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 indicate that the impact of genetic effect becomes more gender-specific during advanced age than at younger ages, and that the genetic 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.