Discussion

Several candidate genes have been explored for their part in human longevity. The majority of the previous studies on the genes investigated here have focused on one or a few variations and have often been followed by contradictory replication results. Interestingly, in this study we did include three of the specific SNPs reported previously (rs4343 (ACE), rs5882 (CETP), and rs1801133 (MTHFR)). Retrospectively inspecting these SNPs in the data shows that only rs4343 (ACE) was nominally significantly associated with longevity (P<0.05) in concordance with the initial publications, while no effect of the other two SNPs could be noted. Thus, these results emphasize that such a single-marker candidate approach might point to a specific polymor- phism, yet it does not enable conclusions on the possible relevance of variation in the gene region as such.

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To facilitate the confirmation of the relevance of a number of well-known candidate genes for longevity, we thus extended the examination of these genes to cover the majority of the common variation in the regions by using a total of 102 tagging SNPs. Apply- – ing the case control approach, two SNPs, rs769449 (APOE) and rs9923854 (CETP), were found to be associated with longevity. The effect of these was supported by haplotype and gene-based analyses and

(a) Danish cohort 1.00 AA+AC CC 0.75 0.50 0.25 0.00 93 98.5 104 Analysis time (age in years) (b) Dutch cohort 1.00 AA AC 0.75 CC 0.50 0.25 0.00 85 90 95 Analysis time (age in years) gnivivrus noitroporPgnivivrus noitroporP

al. 2010). Moreover, in the cohort individuals used in the present paper for investigating the dependency between ε APOE and rs769449, rs429358 alone also shows a modest effect on survival during old age (unpublished data). Yet, such an effect was not seen for rs769449, indicating that rs769449 does not reflect all the effects of rs429358. In contrast, rs9923854 (CETP) did show nominal effects on survival during old age, yet only in females, indicating that the effect of this SNP might become gender specific toward the most late part of life.

Apoe is involved in lipoprotein metabolism via its binding to low-density lipoprotein (LDL) receptors on the surface of for instance liver cells, hereby mediating endocytosis and consequently removal of LDL from the circulation. The isoforms of APOE (E2, E3, and E4) have been shown to interact differently with diverse LDL receptors, leading to differences in LDL levels, APOE4 being associated with the highest LDL levels. Moreover, APOE4 possibly also influences oxidative stress and inflammatory states. These mechanisms are probably the basis of the increased risk of premature ’ atherosclerosis, coronary heart disease, Alzheimer s dis- ε ease, and mortality repeatedly found in APOE 4 car- riers (Chung et al. 2010; Dietrich et al. 2005; Jofre- Monseny et al. 2007; Vitek et al. 2009). The association of rs769449 (APOE) to longevity identified here is probably a proxy for these functional effects.

Fig. 1 Kaplan–Meier survival estimates by rs2069827 (IL6) genotype groups in the a Danish and b Dutch prospective cohorts

was supported in the German replication population, hence pointing to importance of genes involved in lipoprotein metabolism for longevity.

Cetp has opposing effects on lipoprotein metabo- lism. On one hand, it increases LDL and very-low- density lipoproteins (VLDLs) levels by transferring cholesterol esters from high-density lipoproteins (HDLs) to LDLs/VLDLs. On the other hand, it increases removal of lipoproteins from the circulation as part of transferring triglycerides from VLDLs/LDLs to HDLs (reviewed in Weber et al. 2010). Hence, one might expect the rare allele of rs9923854 (CETP) to contribute to decreased cholesterol ester transfer activity and/or increased triglyceride transfer activity, although the role could well be much more complex. This is underlined by the difference in success observed in clinical studies of cetp inhibitors for prevention of atherosclerosis (Weber et al. 2010).

The effect of APOE rs769449 did, however, most ε likely reflect the well-known effect of the APOE 4 haplotype defining rs429358 and was not evident in ε advanced age survival. The APOE 4 haplotype has in our cohorts not only been reported to pose a negative effect on survival from middle age to old age (Gerdes et al. 2000; Tan et al. 2004) but also on survival during old age (Bathum et al. 2006; Deelen et al. 2011; Jacobsen et

CC (N=45) AC (N= 153)182069827 (IL6) genotype groups 

the level of the entire gene indicated a positive effect of the rare variants of HSPA14 on survival from middle-aged to old-aged. HSPA14 belongs to the same functional group as HSPA1A and HSPA1L (70 kDa heat shock proteins (HSP70s)) previously reported as associated with human longevity (Altomare et al.

Fig. 2 Mean age-adjusted ΔC values by rs2069827 (IL6) t genotype groups

Schleswig-K.E.R.N. (by EU funds from the European Regional Development Fund), the National Institute on Aging (P01 AG08761), the Novo Nordisk Foundation, the Aase and Ejnar Danielsen Foundation, the Augustinus Foundation, the Brødrene Hartmann Foundation, the King Christian the 10th foundation, the Einer Willumsens Mindelegat Foundation, and by a Dutch grant from the Netherlands Consortium for Healthy Ageing (grant 050- 060-810) in the framework of the Netherlands Genomics Initia- tive/Netherlands Organization for Scientific Research (NWO). The Danish Aging Research Center is supported by a grant from the VELUX Foundation. Susanne Knudsen,SteenGregersen,Ulla Munk, Shuxia Li, Anne Mette Hedegaard Nielsen, Marlene Graff Sørensen, and Lene Elnegaard are thanked for excellent technical work.