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minimal before age 60 years and most profound from age 85 years and onwards (Hjelmborg et al., 2006). Candidate longevity genes en- code proteins involved in several biological processes including lipid– protein metabolism (APOE being by far the most validated longevity gene to date), growth hormone 1/insulin-like growth factor 1/insulin (GH/IGF-1/INS) signaling, DNA damage signaling, and repair and pro/ antioxidant pathways (Christensen et al., 2006). The genes compos- ing the latter three pathways encode proteins involved in processes such as energy metabolism, growth and differentiation, and the main- tenance of genetic stability (including telomere stability), and protec- tion against free radicals produced by the general metabolism. These are all processes believed to be of key importance for aging and longevity from nematodes to humans (reviewed in e.g. Burtner and Kennedy, 2010; Cluett and Melzer, 2009; Harman, 1973; Kenyon, 2010; Kuningas et al., 2008). Studies using various animal models have found that either depletion or over-expression of some of these candidate genes affect the life span of the organism (e.g. GH, GPX1, FOXO, KL, GHRHR, POLB, RAD52, INS and TP53 (Bartke, 2005; Cabelof et al., 2006; Flurkey et al., 2001; Giannakou et al., 2004; Holzenberger et al., 2003; Kawano et al., 2000; Kuro-o et al., 1997; Kurosu et al., 2005; Park et al., 1999; Tyner et al., 2002; Mockett et al., 1999)), whereas manipulating other genes leads to aging-like phenotypes (e.g. PON1 and CSB (Shih et al., 1998; van der Horst et al., 1997)).

2.1.1. Discovery cohorts

The study population of oldest-old was 1200 participants from The Danish 1905 Birth Cohort Study (1905 cohort) (Nybo et al., 2001). The study was initiated in 1998, when participants were 92.2–93.8 years of age (mean age 93.2). Survivors were re-assessed in 2000, 2003 and 2005. Vital status was followed until death or Jan- uary 1st 2010, whichever came first, resulting in a mean follow-up time for survivors of 11.4 years (range: 11.2–11.6). Survival status in- formation was retrieved from the Danish Central Person Register, which is continuously updated (Pedersen et al., 2006). The younger control group (46.0–55.0 years of age, mean age 50.5) was 800 indi- viduals from The Study of Middle Aged Danish Twins (MADT) (Skytthe et al., 2002). MADT was initiated in 1998 by random selec- tion of 2640 intact twin pairs from 22 consecutive birth years (1931–1952) via the Danish Central Person Register. Only one twin from each twin pair was included in the present study. Permission to collect blood samples and usage of register based information was granted by The Danish National Committee on Biomedical Re- search Ethics for both surveys. All participants gave their informed consent.

After data cleaning, the final sample size for data analysis was 1089 oldest-old and 736 middle-aged individuals. Of these 1089 oldest-old, 14 were alive by January 1st 2010, hence, for the longitu- dinal analysis their remaining life expectancies were imputed using www.mortality.org, which, by appliance of data from the Statistics Denmark, contains cohort mortality data for the Danish population. Of the 736 MADT individuals, 35 had died since 1998. Based on period life table data for the Danish population (www.mortality.org) it is an- ticipated that only approximately 9% of the remaining 701 individuals will become 93 years of age.

In humans rare mutations that severely affect the function of some of these genes (WRN, CSB and RECQL4) can cause segmental progeroid premature aging syndromes such as Werner's, Cockayne's and Rothmund–Thomson syndromes (reviewed in Burtner and Kennedy, 2010)), whereas more common genetic variation has been reported to be associated with human lifespan. Examples from the GH/IGF-1/INS pathway include IGF1R, GHR and FOXO3A, from the DNA damage signaling and repair pathway WRN, MLH1 and TP53 and from the pro/antioxidant pathway SOD1, SOD2 and PON1 (Bonafe et al., 2003; Castro et al., 2000; Christiansen et al., 2004; Flachsbart et al., 2009a; Kim et al., 2006; van Heemst et al., 2005a; van Heemst et al., 2005b; Willcox et al., 2008a; Yashin et al., 2000).

2.1.2. Replication samples

For replication of findings from the Danish case–control study, two German samples of 1613 unrelated long-lived individuals (age 95–110) and 1104 middle-aged controls (mean age 67.2) were used. The participants were identified via the local registry offices and the Federal Administrative Office. They were recruited from dif- ferent geographic regions of Germany and were all of German ances- try (Nebel et al., 2005). Approval was received from the Ethics Committee of the University Hospital Schleswig-Holstein and from local data protection authorities. All participants gave their informed consent.

The vast majority of the candidate genetic association studies pub- lished so far focus on a single or a few single nucleotide polymor- phisms (SNPs) or candidate genes, although recently a few studies have initiated the investigation of SNPs in groups of candidate genes and pathways based on the functional roles of the gene products (Deelen et al., 2011; Flachsbart et al., 2010; Nebel et al., 2009; Pawlikowska et al., 2009a). In addition, previous studies have gener- ally applied a case–control study design, thus raising concerns of bias introduced by differences in characteristics of cases and controls, for example cohort effects.