Gene Polymorphism RR (95% CI) females RR (95% CI) males GHRHR codon 57 SNP 0.90 (0.66–1.21) 1.24 (0.82–1.87) GH1 intron SNP 0.80 (0.67–0.96) 1.14 (0.88–1.48) IGF1 CA repeat 0.85 (0.71–1.01) 1.01 (0.79–1.30) CT repeat 1.23 (0.94–1.60) 0.78 (0.53–1.16) INS VNTR 1.05 (0.88–1.25) 0.99 (0.77–1.27) IRS1 codon 972 SNP 0.80 (0.62–1.03) 0.95 (0.70–1.30)

The analysis of the combined cohorts of the Leiden 85-plus Study contained 866 females and 379 males. Bold: significant result. The polymorphisms were analysed as independent covariates in a left-censored Cox proportional hazards model.

The polymorphisms were analysed as independent covariates in a linear regression model. Bold: significant result.

five selected polymorphisms that lead to reduced IIS activity (GH1 SNP, IGF1 CA repeat and IRS1 SNP) and reached statistical significance for the GH1 polymorphism (Table 3). Our data show that mortality risk (95% confidence interval) is 0.80 (0.67– 0.96) reduced (P = 0.019) for female carriers of the variant GH1 allele compared with non-carriers, with risk estimates being sim- ilar in the two independent cohorts [0.79 (0.63–0.99) and 0.86 (0.62–1.20), respectively, for cohorts ’87 and ’97].

became apparent (Table 5). Lower composite IIS6 scores were associated with lower old age mortality in women (P for linear trend = 0.045), but not in men.

Assigning one point to each high-risk allele and subtracting one point for each low-risk allele is arbitrary as it ignores obvious and pertinent complications (dominance, epistasis, gene-by- environment interactions among many others). However, we have deliberately chosen the simplest model because the nec- essary biological information on IIS pathway variants in humans to build more sophisticated models is currently lacking. The results depicted in Table 3 suggest, however, that we may have underestimated the effects of the composite IIS6 score because in females the polymorphisms in GH1, IGF1 CA and IRS1 seemed to contribute most to longevity, although the effect estimates for these three polymorphisms were similar. We therefore also calculated a composite IIS3 score, based on only these three polymorphisms. Within the IIS3 score categories, we detected a highly significant survival benefit in females for the lower IIS3 scores compared with the reference category (IIS3 score 0), the relative mortality risks being 0.92 (0.72–1.18) for IIS3 score −1, 0.74 (0.58–0.96) for IIS3 score −2 and 0.46 (0.27–0.79) for IIS3 score −3, suggesting that the beneficial effects of these three polymorphisms on survival in females

The next question we addressed was whether the observed effects were additive. To do this, we calculated a composite IIS6 score, by assigning a value of −1 for carriership of each variant predicted to cause reduced IIS activity (GHRHR, GH1, IGF1 CA, IGF1 CT, INS and IRS1) and a value of +1 for carriership of the variant predicted to cause increased IIS activity (IGF1 CT). We then determined the relationship between composite IIS6 score and body height for both sexes in cohort ’97 of the Leiden 85- plus Study. It was expected that lower IIS6 scores would asso- ciate with lower body height, and we observed this trend for both men and women (Table 4). The result was statistically significant for women, at P = 0.004 when using IIS6 score as a continuous variable and P = 0.029 when using IIS6 score as a categorical variable, not assuming a linear additive effect. We then determined the relationship between composite IIS6 score and survival for both sexes in the combined cohorts of the Leiden 85-plus Study. In this analysis again a sex difference

Females (n = 298) Males (n = 156) IIS6 score n Body height (SD) ∆ n Body height (SD) ∆ +1 0 – – 0 – – 0 21 157.4 (6.3) 0 8 169.8 (6.0) 0 −1 75 157.8 (6.2) +0.4 37 168.6 (6.7) −1.2 −2 113 155.9 (6.3) −1.5 60 168.7 (5.5) −1.1 −3 64 154.2 (6.1) −3.2 36 167.7 (8.3) −2.1 −4 24 155.7 (6.0) −1.7 14 167.0 (6.1) −2.8 −5 1 153.5 −3.9 1 166.5 −3.3 P for linear trend 0.004 0.24

Table 4 Association between composite IIS6 and body height at age 85 years in cohort ’97 of the Leiden 85-plus Study

∆, difference in mean body height compared with reference category IIS6 score = 0. Body height is expressed in centimetres. SD, standard deviation. Bold: significant result.

Females (n = 704) Males (n = 325) IIS6 score n RR 95% CI n RR 95% CI +1 6 1.5 0.59–3.8 1 13 1.7–102 0 57 1 27 1 −1 186 0.89 0.63–1.3 84 1.26 0.77–2.1 −2 270 0.84 0.60–1.2 120 1.43 0.89–2.3 −3 155 0.78 0.54–1.1 76 1.28 0.78–2.1 −4 29 0.69 0.39–1.2 16 0.83 0.36–1.9 −5 1 – – 1 – – P for linear trend 0.047 0.90

RR, relative mortality risk as estimated in a left-censored Cox proportional hazards model. Bold: result.

RR, relative mortality risk as estimated in a left-censored Cox proportional hazards model. Bold: significant

at large that carriers of combinations of variants that are known to result in decreased IIS signalling are associated with reduced body height and improved old age survival, the effects on sur- vival, however, being confined to women. Of all polymorphisms analysed, these results were most significant for the GH1 SNP, as female carriers of the GH1 SNP were significantly smaller and exhibited a significant benefit in old age survival.

Table 6 Relative mortality risks (RR) for carriers of at least one variant allele compared with non-carriers for the polymorphisms selected from the oxidative stress response as determined in the combined cohorts of the Leiden 85-plus Study

RR (95% CI) RR (95% CI) Gene Polymorphism females males SOD2 exon 16 SNP 1.16 (0.95–1.42) 0.90 (0.68–1.20) UCP2 promoter SNP 1.06 (0.86–1.30) 1.04 (0.79–1.38) UCP2 45-bp insertion/deletion 0.93 (0.76–1.14) 0.84 (0.65–1.10)