e-mail: D.van_Heemst@lumc.nl

Accepted for publication 19 January 2005

Data from other mutant mice models support a link between longevity and the IGF-1 branch of the IIS pathway. The IGF-1 branch acts through growth-hormone-releasing hormone (GHRH) produced by the hypothalamus, GH produced by the anterior pituitary and IGF-1 produced by the liver and peripheral tissues to control mammalian body size. Four dwarf mice models with impeded IGF-1 production, Prop1df/df (Brown-Borg et al., 1996), Pit1dw/dw (Flurkey et al., 2001), GHRHRlit/lit (Flurkey et al., 2001) and GHR–/– (Chandrashekar et al., 1999), all show a long- lived phenotype. However, these four mice models show other hormonal abnormalities besides IGF-1 deficiency (Carter et al., 2002). Recent results obtained with mice mutated for the IGF- 1 receptor hint at a direct role for reduced IGF-1 signalling in mammalian longevity: Igf1r+/– females, but not males, exhibit a long-lived phenotype (Holzenberger et al., 2003). Interestingly, the long-lived igf1r+/– females show reduced activation of p66Shc and increased resistance to oxidative stress. Possibly, the life extension of igfr1+/– females occurs through the same mechanism as in long-lived p66shc1–/– mice (Migliaccio et al., 1999), in which forkhead transcription activity is up-regulated and oxidative stress resistance is increased (Nemoto & Finkel,

Results

For all analysed polymorphisms, genotyping error rates were below 1% and the observed genotype distributions were in Hardy–Weinberg equilibrium. The linkage disequilibrium for the two polymorphisms in the IGF1 gene and the UCP2 gene was relatively weak (respectively D′ = 0.12 and D′ = 0.46). More- over, none of the observed allele frequencies was statistically different between cohort ’87 and cohort ’97 (Table 1). We had selected six polymorphisms from the IIS pathway based on pre- vious reports of their effects on gene expression or protein func- tion. Here, we wanted to determine their effect on two complex phenotypes in the Leiden 85-plus Study, namely body height and longevity. Because we were unaware of the relative impact on each of the two phenotypes (body height and longevity) of the genetic variations selected from the human IIS pathway, nor of their relationship, we have chosen first to analyse them as independent covariates in a linear regression model (for body height) and a left-censored Cox proportional hazards model (for longevity). Because, in Drosophila and mice, effects of IIS variants were often more pronounced in females than in males, we per- formed these analyses stratified for gender.

In both males and females, except for the IGF1 CT repeat, the polymorphisms leading to reduced IIS associated with reduced body height, whereas the polymorphism leading to increased IIS associated with increased body height in cohort ’97 of the Leiden 85-plus Study. Generally these effects were small and did not reach statistical significance except for results for the GH1 polymorphism in females (Table 2). Body height was 2.0 cm lower for female carriers of the variant GH1 allele

seems evolutionarily conserved from worms to rodents, with effects on longevity often being stronger in the female sex. In all species analysed, most long-lived IIS mutants also show increased resistance to oxidative stress. Since the discovery of the role of the IIS pathway in longevity in model organisms researchers have speculated on the potential effects of muta- tions in this pathway in humans. Only few human data have been reported (Bonafe et al., 2003). Here we report data on the relationship between variation in several components of the IIS pathway and prospective survival in a well-studied Dutch population of elderly people. The IIS pathway genes studied include several components from the evolutionarily complex (or

We determined the effect of each of these polymorphisms on old age mortality in the combined cohorts of the Leiden 85- plus Study. Here, we observed different and often opposite effects for males and females. In females, the relative mortality risks for carriers of the variant allele were lower for three of the

Cohort ’87 Cohort ’97 Combined cohorts Enrolled (n) 977 599 1576 Birth cohort 1883−1902 1912−1914 1883−1914 Median age baseline (range) 89.1 (85.0–103.6) 85.0 (85.0–85.0) 87.1 (85.0–103.6) Females/males (n) 710/267 397/202 1107/469 DNA available (n) 682 563 1245 GHRHR, minor allele 0.053 0.045 0.049 GH1, minor allele 0.411 0.422 0.416 IGF1 CA, sum variant alleles 0.330 0.348 0.339 IGF1 CT, 192-bp allele 0.062 0.069 0.065 INS, class III allele 0.303 0.275 0.291 IRS1, minor allele 0.072 0.072 0.072 SOD2, minor allele 0.494 0.508 0.500 UCP2, minor allele promoter 0.372 0.406 0.388 UCP2, 45-bp insertion 0.275 0.302 0.288

GHRHR = Growth Hormone Releasing Hormone Receptor, GH1 = Growth Hormone 1, IGF1 = Growth Factor 1, INS = Insulin, IRS1 = Insulin Receptor Substrate 1, SOD2 = Superoxide Dismutase 2, UCP2 = Uncoupling Protein 2, CA = CA repeat, CT = CT repeat.

Table 2 Difference in body height at age 85 years between carriers of at least one variant allele compared with non-carriers for the selected IIS polymorphisms as determined in cohort ’97 of the Leiden 85-plus Study

Table 3 Relative mortality risks (RR) for carriers of at least one variant allele compared with non-carriers for the selected IIS polymorphisms as determined in the combined cohorts of the Leiden 85-plus Study

Gene Polymorphism Females (n = 298) Males (n = 156) GHRHR codon 57 SNP +1.0 cm (P = 0.409) +0.7 cm (P = 0.715) GH1 intron SNP −−−−2.0 cm (P = 0.007) −1.9 cm (P = 0.078) IGF1 CA repeat −0.9 cm (P = 0.180) −0.4 cm (P = 0.693) CT repeat −0.8 cm (P = 0.415) +1.4 cm (P = 0.375) INS VNTR −0.5 cm (P = 0.358) −0.6 cm (P = 0.462) IRS1 codon 972 SNP −0.4 cm (P = 0.706) −1.7 cm (P = 0.183)