Of the polymorphisms analysed, the association with body height and longevity is most significant for the GH1 SNP (in female carriers), although a similar trend was observed for two of the other polymorphisms tested (the IGF1 CA repeat and the IRS1 SNP). This could be caused by differences in the effects of the genetic variants on gene expression or protein function or by differences in the impact of these effects. Because the IIS pathway is a hierarchical network, effects of variation in IIS com- ponents may depend on their position within the hierarchy. We speculate that effects of variation in regulatory components that arose relatively late in evolution, such as GH1, may be of greater importance for human lifespan than variation in core compo-

In conclusion, we present evidence that in females but not in males reduced IIS activity is associated with longevity. Of the analysed polymorphisms, results were most pronounced for the GH1 SNP. The detected associations with longevity are in line with corresponding associations with body height, and the observed sex difference can be explained by biologically determined sex differences in the regulation of the GH/IGF-1

One of the mechanisms postulated for the life-extending effect of reduced signalling via the GH/IGF-1 axis is increased resistance to oxidative stress. In mice, administration of GH or IGF-1 in vitro as well as in vivo significantly suppresses enzymes implicated in the antioxidative stress response (Brown-Borg & Rakoczy, 2003). We therefore looked at the effect of polymor- phisms in genes implicated in the antioxidative stress response on longevity. One mechanism by which forkhead transcription factor FOXO3a (also known as FKHR-L1), the closest mammalian homologue of C. elegans DAF-16, protects quiescent cells from oxidative stress is increasing the expression of manganese superoxide dismutase (SOD2) (Kops et al., 2002), which con- verts superoxide to hydrogen peroxide. Mild uncoupling of oxi- dative phosphorylation that decreases mitochondrial reactive oxygen species (ROS) production through up-regulation of uncoupling proteins, such as UCP2 (Walder et al., 1998), is another part of the antioxidative stress response. However, none of the tested polymorphisms in SOD2 or UCP2 was asso- ciated with lifespan in our analyses. This result is in line with recent data from C. elegans (Murphy et al., 2003). In C. elegans, multiple effector genes are coordinately regulated by DAF-16 but probably act in a cumulative manner to influence lifespan, as they were found to have only small effects on lifespan by themselves when compared with the effect of DAF-16. In our analyses in humans, where the effects of key components from the IIS pathway on longevity being modest, the influence of downstream effector genes may be extremely difficult to detect. Moreover, this analysis does not rule out the possibility that the

Experimental procedures

Subjects

For the prospective follow-up study, we made use of the population-based Leiden 85-plus Study (Table 1), which consists of two cohorts of inhabitants of Leiden, The Netherlands (Slagboom et al., 2000; Bootsma-van der Wiel et al., 2002). For cohort ’87, inhabitants of Leiden aged 85 years and over were enrolled between 1987 and 1989 and followed for mortality until May 2001. For cohort ’97, inhabitants of Leiden of exactly 85 years were enrolled between 1997 and 1999 and followed for mortality until April 2004. There were no exclusion criteria related to health. In cohort ’87, almost 14 years after the start of the study 704 had died (98%). In cohort ’97, almost 7 years after the start of the study 308 had died (51%). Body height was measured using a tape measure for participants of cohort ’97 at age 85 years when standing upright without shoes.

The Leiden 85-plus Study was approved by the medical ethics committee of the Leiden University Medical Centre. Informed consent was obtained from all participants after the nature and possible consequences of the studies were explained.

measurements on an ABI7900 (both Applied Biosystems). The IGF1 CA repeat (Weber & May, 1989), the IGF1 CT repeat (Arends et al., 2002) and a 45-bp insertion/deletion polymor- phism in UCP2 (Esterbauer et al., 2001) were analysed by frag- ment analysis using a ABI3700 (Applied Biosystems) as described. For determination of the INS VNTR, all samples were genotyped for the −23/HphI restriction fragment polymorphism, as this polymorphism is in very tight linkage disequilibrium with INS VNTR class alleles in populations of European descent (99.6% concordance between −23/HphI and INS VNTR alleles) (Bennett & Todd, 1996). As a quality check, for each polymor- phism, 10% of the samples were genotyped twice.

Selected polymorphisms

On the basis of sequence and/or functional homology with components of the IIS pathway and/or oxidative stress response pathway that were associated with longevity in C. elegans, D. melanogaster and/or mice, we constructed a list of human candidate genes from these signalling pathways. We next searched PubMed and dbSNP for variations in these candidate genes occurring at a frequency of at least 5% that were pre- viously associated with changes in the expression level of the gene and/or the activity of the encoded protein. Such polymor- phims were found for GHRHR, GH1, IGF1, INS and IRS1 (IIS path- way; see supplementary Table S1) and SOD2 and UCP2 (oxidative stress response; see supplementary Table S2).

Supplementary material

The following tables are available as supplementary material at http://www.blackwellpublishing.com/products/journals/suppmat/ ACE/ACE148/ACE148sm.htm

Statistical analyses

Linear regression was used to model the relation of genotypes with body height.

Table 1 Polymorphisms from the IIS pathway selected on the basis of previously described functionality and their expected effect on IIS activity.