Previously, rs429358 had been associated with several meta- bolic phenotypes, such as ApoE, total cholesterol, HDL choles- terol, LDL cholesterol, and hsCRP levels, as well as HDL and LDL particle size (Boerwinkle & Utermann, 1988; Topic et al., 2008; Hubacek et al., 2010) and, here, we have confirmed these find- ings using serum measurements of the offspring and partners from the LLS. Because the insulin⁄IGF-1 signaling (ISS) pathway has a lifespan regulating effect in several model organisms (Fon- tana et al., 2010; Kenyon, 2010) and humans (Suh et al., 2008), we also investigated the effect of rs429358 on serum levels of IGF-1 and IGFBP3, which both play a role in this pathway. Both proteins are involved in the etiology of several age-related dis- eases. However, up till now, it is not clear whether higher or lower serum levels are beneficial for longevity. Low IGF-1 serum levels associate to a decreased risk of cancer, but an increased risk of cardiovascular disease and neurodegenerative disease (Juul, 2003). Previously, we showed in the Leiden 85-Plus Study cohort that genetic variants known to associate to lower IIS activity and IGF-1 serum levels at younger age associated with better survival at ages above 85 years (van Heemst et al., 2005). However, the effect of these genetic variants on IGF-1 serum levels was not tested in the Leiden 85-Plus Study cohort. In addi- tion, we showed previously that neither IGF-1 and IGFBP3 levels nor their ratio differed between partners and offspring from the LLS (Rozing et al., 2009), which indicates that IGF-1 serum levels are, in middle age, not a marker for longevity, whereas a decreased risk of metabolic diseases was evident at that age in long-lived families (Westendorp et al., 2009). In the current study, we found that the minor allele of rs429358 associates with lower IGF-1 levels in middle-aged women, which to our knowledge has not previously been reported. Like low IGF-1 lev- els, ApoE e4 was previously associated with an increased risk of developing cardiovascular disease and neurodegenerative dis- ease (Corder et al., 1993; Eichner et al., 1993; Christensen et al., 2006). Thus, the mechanism behind the increased risk of female ApoE e4 carriers of developing cardiovascular and⁄or neurodegenerative diseases might involve serum levels of IGF-1 or other aspects of IIS activity reflected by these levels. Apart from lipid metabolism, the parameters determining the longev- ity phenotype in middle age in the LLS, such as glucose metabo- lism, insulin sensitivity, and thyroid hormone metabolism (Rozing et al., 2009, 2010a,b; Vaarhorst et al., 2011; Wijsman et al., 2011), were not influenced by the presence of the minor allele of rs429358. This indicates that it is likely that other loci could explain the differences in these phenotypes between LLS offspring and partners. ª 2011 The Authors Aging Cell ª 2011 Blackwell Publishing Ltd/Anatomical Society of Great

same number of disease risk alleles for cardiovascular disease, cancer, and type 2 diabetes as young controls (Beekman et al., 2010), we expected to primarily find longevity-promoting alleles. However, although most of the 58 prioritized SNPs (n = 43) from the LLS GWAS showed a longevity-promoting effect ranging from 36 to 168%, none of them could be repli- cated in additional study populations of nonagenarian single- tons. The only replicated locus is APOE, which is a mortality locus that has previously been reported to be the major locus responsible for Alzheimer’s disease (Harold et al., 2009; Lambert et al., 2009; Seshadri et al., 2010), a well-known age- related disease. Nevertheless, none of the other Alzheimer’s disease loci showed an association with survival to 90 years, which indicates that the remaining genetic variation in longevity in the LLS could not be explained by the genetic variation which contributes to Alzheimer’s disease. In addition to APOE, we also observed evidence for association at the previously reported AKT1 locus (Pawlikowska et al., 2009) with survival into old age in the LLS GWAS, although the effect of this SNP is relatively small (25% decreased probability of becoming 90 years) com- pared to the effect of rs429358 (51%). The previously reported longevity-promoting effect of the FOXO3A locus could not be replicated in this study. This is probably due to the relatively low number of centenarians in the LLS GWAS case group, in which the effect of SNPs in FOXO3A on longevity seems to be most prominent. The still unexplained genetic variation in longevity might be attributable to rare variants or variants with small effects, which has previously been reported for other complex traits, such as Alzheimer’s disease. These loci could not be iden- tified in this study because of the relatively small number of cases in the LLS GWAS, the heterogeneity of factors influencing lifespan within populations, and the difference in the design of the studies used for replication. One way to identify variants with small effects would be to increase the initial sample size of the GWAS study and perform replication in other studies of nonagenarians. Given the higher heritability of longevity at older ages (Tan et al., 2004), one may also limit the study population

and 1989, 673 subjects aged 85 years and older were enrolled in a prospective study (Cohort 1). Between 1997 and 1999, 563 subjects were enrolled in the month of their 85th birthday with follow-up (Cohort 2). Subjects were visited at their home, and there were no exclusion criteria related to health. DNA was available from the combined cohorts consisting of 1208 subjects aged 85 years and older (Leiden 85-plus replication cases).

variants with small effects, rare variants with large effects might play a role in longevity. By whole-genome⁄exome sequencing of long-lived subjects and their families, rare variants can be identified and associated with human longevity.