The insulin/insulin-like growth factor (IGF-1) signaling (IIS) pathway is considered as a candi- date pathway for studying human longevity. It is involved in the adaptation of the organism to its (changing) environment (Tatar et al. 2003). When experimentally induced in model organisms like worms, flies, and mice, mutations in genes that play a role in IIS, e.g., homologues of human IGF1R, INSR, IRS1, PI3K, and FOXO, were shown to have a considerable effect on lifespan (Kenyon et al. 1993; Kimura et al. 1997; Tatar et al. 2001; Holzenberger et al. 2003; Bluher et al. 2003; Morris et al. 1996; Friedman and Johnson 1988; Clancy et al. 2001; Hwangbo et al. 2004; Ogg et al. 1997; Lin et al. 1997; Giannakou et al. 2004; Selman et al. 2011). Although the IIS pathway is evolutionarily conserved, the complexity of the human IIS pathway (Fig. 1) is much larger compared to that of model organisms.

Since the amount of tests in SNP set analysis is low compared to single SNP analysis, it requires a lower penalty for multiple testing. Therefore, SNP set analysis is also very suitable in studies with low power for GWAS analysis. The last couple of



Fig. 1 Insulin/IGF-1 signaling pathway. The insulin/IGF-1 signaling pathway consists of the core components IGF1R/IR/ IRR, IRS, PI3K, AKT/SGK, FOXO and SIRT, and proteins that have a direct activating or inhibiting effect on these proteins. The small closed circles (containing Ac, P, or Ub) indicate an activating effect of the posttranslational modification on the

protein, while the small dashed circles indicate an inhibiting effect. The straight arrows pointing to these small circles indicate an activating effect on the posttranslational modifica- tion, while the dashed arrows indicate an inhibiting effect. Ac acetylation, P phosphorylation, Ub ubiquitylation

Several studies have investigated associations be- tween single SNPs in genes from the IIS pathway and human longevity. The most prominent results came from FOXO3 (Willcox et al. 2008; Flachsbart et al. 2009; Anselmi et al. 2009; Pawlikowska et al. 2009; Li et al. 2009; Soerensen et al. 2010) and AKT1 (Pawlikowska et al. 2009), which showed associa- tions with longevity in several independent cohort studies.

showed contradictory results (Martin-Ruiz et al. 2005; Bischoff et al. 2006). Telomere integrity is essentially regulated by two protein networks, telomerase and its associated factors, which regulate telomere length, and the shelterin complex, which covers the telomeres (de Lange 2005; Collins and Mitchell 2002) (Fig. 2). Several studies have investigated associations be- tween single SNPs in telomerase and shelterin genes and telomere length. The most promising results came from TERC and TERT (Atzmon et al. 2010; Codd et al. 2010; Levy et al. 2010; Mirabello et al. 2010; Rafnar et al. 2009), of which the latter has also been associated with human longevity (Atzmon et al. 2010). In this study, we used four self-contained tests (PLINK set-based test, Purcell et al. 2007; GRASS, Chen et al. 2010; Global test, Goeman et al. 2004; and SNP ratio test, O'Dushlaine et al. 2009) and one competitive test (the comparative approach of Global test) to study the joint effect of genetic variation in the IIS and TM pathways on human longevity. For the analyses, we used genotyped GWAS data of nonage- narian siblings from the Leiden Longevity Study (LLS) and younger population controls from the

Another candidate pathway for studying human longevity is the mechanism of telomere maintenance (TM). Telomeres are structures at the end of chromo- somes, consisting of TTAGGG tandem repeats (Moyzis et al. 1988), which protect chromosomes from degradation or rearrangement (Blackburn 1991). In normal human cells, telomere length declines with every cell division (Harley et al. 1990), and when a critical length is reached, the cell will enter replicative senescence (Allsopp 1996). In human epidemiological studies in blood, increased telomere length has been associated with longevity (Atzmon et al. 2010), while decreased telomere length has been associated with increased mortality (Cawthon et al. 2003; Bakaysa et al. 2007; Kimura et al. 2008), although some studies

associated with human longevity (Atzmon et al. 2010). In this study, we used four self-contained tests (PLINK set-based test, Purcell et al. 2007; GRASS, Chen et al. 2010; Global test, Goeman et al. 2004; and SNP ratio test, O'Dushlaine et al. 2009) and one competitive test (the comparative approach of Global test) to study the joint effect of genetic variation in the IIS and TM pathways on human longevity. For the analyses, we used genotyped GWAS data of nonage- narian siblings from the Leiden Longevity Study (LLS) and younger population controls from the Rotterdam Study (RS) (Deelen et al. 2011).

a b

ophthalmological diseases (Teichert et al. 2009). The study consists of 7,983 participants from the baseline cohort (RS-I) and 3,011 participants from an inde- pendent extended cohort formed in 1999 (RS-II) from which DNA was isolated between 1990 and 1993 (RS-I) or between 2000 and 2001 (RS-II). For the GWAS, 1,731 participants from the combined cohort who were below 60 years of age and for whom GWAS data were available were included as controls (LLS GWAS controls) (Deelen et al. 2011).

Population substructure

Fig. 2 Telomere maintenance pathway. The telomere mainte- nance pathway consists of proteins belonging to telomerase and its associated factors or to the shelterin complex. Telomere elongation is performed by telomerase after binding to the telomere (a). However, binding of the shelterin protein POT1 to the telomere blocks this process (b)

Multidimensional scaling analysis in PLINK (http:// pngu.mgh.harvard.edu/purcell/plink, Purcell et al. 2007) showed that there was no substructure in the GWAS data to an extent that would affect the observations (Deelen et al. 2011).

Materials and methods

Genotyping and SNP selection