would explain the scattered association in the IIS pathway.

abnormalities in growth and/or increased mortality (www.informatics.jax.org; Blake et al. 2011), which indicates that these genes are indeed responsible for the growth- and lifespan-regulating effects of the IIS pathway. Previously, SNPs in several of the signifi- cant IIS pathway genes (AKT1, FOXO4, INS, and PIK3CA) were studied by single SNP analysis, and only one SNP, rs3803304 in AKT1, which was not measured in our study, showed an association with longevity (Pawlikowska et al. 2009). However, gene set testing, which could have detected association of additional genes containing SNPs with many small effects, was not applied in that study. Most signaling cascades require cooperation of several genes in multiple branches of the cascade. This indicates that, for signaling pathways, mutations in different genes could result in similar downstream effects, which

Although SNPs in FOXO3A have previously associated with longevity in several independent (Willcox et al. 2008; Flachsbart et al. 2009; Anselmi al. 2009; Pawlikowska et al. 2009; Li et al. Soerensen et al. 2010), the gene SNP set showed effect in our study in the PLINK set-based test, test, and SNP ratio test (P=0.181, P=0.138, and P= 0.180, respectively) (Table 5). This might be due the fact that the effects of FOXO3A on longevity most prominent in centenarians. As was reported by Flachsbart et al., centenarians represent highly selected phenotype even among (Flachsbart et al. 2009). In addition, the contribution to longevity in general is increased at ages (Hjelmborg et al. 2006), and the small effects longevity-promoting gene variants, relative to factors, may be larger in centenarians (Perls et al. and not detectable in nonagenarians. The cases our study, which are from long-lived have a mean age of 94 years, yet we had only individuals >100 years, which may explain the of significance of the FOXO3A association in population.

Table 4 Results of gene set analysis of insulin/IGF-1 signal- ing, telomere maintenance, and eye and hair color pathway SNP sets

Pathway test Insulin/IGF-1 Telomere Eye and signaling maintenance hair color PLINK set-based testa 0.064 0.019 0.340 GRASSa 0.010 0.023 0.540 Global testa 0.011 0.023 0.362 SNP ratio testa 0.044 0.034 0.337

POT1 is part of the shelterin complex and is responsible for the binding of this complex to the TTAGGG repeats of telomeres. Binding of POT1 to the telomere leads to decreased elongation by telo- merase (de Lange 2005). Reduction of POT1 in human fibroblasts by RNAi leads to induction of

aPermutation (n=10,000) P value

Table 5 (continued) Gene PLINK set- Global testa SNP based testa ratio PIK3R2 1 0.722 1 PIK3R3 1 0.263 1 PPP2R5B 1 0.363 1 PRR5 0.355 0.163 0.257 PTEN 1 0.855 1 PTPN1 1 0.982 1 RBL2 1 0.061 1 RICTOR 1 0.343 1 SCP2 1 0.729 1 SGK1 0.091 0.007 0.016 SGK2 0.027 0.042 0.349 SIRT1 1 0.941 1 SIRT2 1 0.282 1 SIRT3 0.241 0.232 0.326 SKP2 1 0.898 1 SOCS1 1 0.349 1 SOCS3 1 0.996 1 SOD2 1 0.692 1 USP7 0.025 0.101 0.103 YWHAB 1 0.223 1 YWHAE 0.067 0.124 0.196 YWHAG 0.090 0.032 0.018 YWHAH 1 0.236 1 YWHAQ 0.228 0.175 0.293 YWHAZ 1 0.756 1

Table 5 Results of gene set analysis of insulin/IGF-1 signaling pathway gene SNP sets

gene SNP sets Gene PLINK set- PLINK set- Global testa SNP based testa based testa ratio testa PIK3R2 1 0.003 0.002 0.099 PIK3R3 1 0.193 0.461 0.197 PPP2R5B 1 0.101 0.023 0.043 PRR5 0.355 1 0.678 1 PTEN 1 1 0.539 1 PTPN1 1 1 0.661 1 RBL2 1 1 0.471 1 RICTOR 1 0.248 0.073 0.073 SCP2 1 1 0.528 1 SGK1 0.091 1 0.675 1 SGK2 0.027 1 0.495 1 SIRT1 1 1 0.378 1 SIRT2 1 1 0.823 1 SIRT3 0.241 1 0.219 1 SKP2 1 1 0.688 1 SOCS1 1 0.181 0.138 0.180 SOCS3 1 0.023 0.023 0.055 SOD2 1 0.156 0.172 0.173 USP7 0.025 0.342 0.042 0.148 YWHAB 1 0.054 0.373 0.491 YWHAE 0.067 0.028 0.019 0.084 YWHAG 0.090 0.022 0.049 0.188 YWHAH 1 0.154 0.217 0.286 YWHAQ 0.228 0.139 0.247 0.224 YWHAZ 1 1 0.873 1 aPermutation (n=10,000) P value 1 0.569 1 1 0.605 1 1 0.905 1 apoptosis, chromosomal 1 0.248 1 (Yang et al. 2005). The same 1 0.132 1 Pot1b knockout mice (He et 0.185 0.531 0.215 al. 2008). In addition, 1 0.198 1 knockout mice show a 0.191 0.885 0.068 “ ” to normal 1 0.372 1 et al. 2008), which stresses 1 0.593 1 lifespan regulation. Most 1 0.722 1 one or several proteins 1 0.547 1 of the complex, e.g., binding, 0.113 0.398 0.200 repression activity. This 1 0.364 1 containing a protein complex, 0.003 9:36  10(cid:3)4 0.022 gene, encoding such an 1 0.726 1 sufficient to alter the function 1 0.828 1 would explain the single-gene 1 0.666 1 pathway.

aPermutation (n=10,000) P value

(Yang et al. 2005). The same effects are observed in Pot1b knockout mice (He et al. 2009; Hockemeyer et al. 2008). In addition, telomerase-deficient Pot1b knockout mice show a reduction in lifespan compared “ ” to normal telomerase-deficient mice (Hockemeyer et al. 2008), which stresses the importance of TM in lifespan regulation. Most protein complexes contain one or several proteins essential for specific functions of the complex, e.g., binding, transport, or activation/ repression activity. This indicates that, for pathways containing a protein complex, mutations in a single gene, encoding such an essential protein, could be sufficient to alter the function of the complex, which would explain the single-gene association in the TM pathway.

Table 6 Results of gene set analysis of telomere maintenance pathway gene SNP sets

Gene PLINK set-based testa Global testa SNP ratio testa ACD 1 0.491 1 DKC1 1 0.642 1 GAR1 1 0.281 1 NHP2 1 0.759 1 NOP10 1 0.208 1 POT1 0.007 0.014 0.019 TEP1 1 0.525 1 TERC 1 0.202 1 TERF1 1 0.821 1 TERF2 0.018 0.160 0.164 TERF2IP 1 0.825 1 TERT 1 0.471 1 TINF2 1 0.587 1

aPermutation (n=10,000) P value