Acknowledgments:

We are grateful to J. J. Houwing-Duistermaat for the use of multivariate analysis. We thank all other members of the Long Life study group: A. Ayrinhac, E. A. Baldal, G. J. D. I. Boomsma, P. M. Brakefield, R. Bijlsma, J. C. van Ingen, D. L. Knook, I. Meulenbelt, P. H. E. M. de Meijer, and J. L. P. W. G. M. van de Zande.

We also thank C. de M. van Schie and M. van Kersbergen-van Oostrom for their assistance.

We are grateful to Gerrit van der Steege and Bruinenberg for the use of their ABI7900 Taqman equipment in Groningen.

This work was funded by an IOP (Innovative Research) grant from the Dutch Ministry of Economic Affairs (grant number IGE01014).

To test for combined effects of the selected polymorphisms, a composite (sub)pathway score was calculated for each participant by assigning a score of +1 for carriership of a variant causing increased signaling and a score of 1 for carriership of each variant causing decreased signaling.

Alternatively, the polymorphisms belonging to the same (sub)pathway (IIS pathway or oxidative stress response) were tested simultaneously as independent covariates in the above-mentioned models.

We used multivariate analyses to adjust for the effects of other functional variants from the same pathway.

SPSS and STATA were used for the statistical analyses, and Thesias was used for the calculation of linkage disequilibrium between the two polymorphisms in IGF1 and the two polymorphisms in UCP2.

References:

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Bartke A, Chandrashekar V, Dominici F, Turyn D, Kinney B, Steger R, Kopchick JJ (2003) Insulin-like growth factor (IGF-) and aging: controversies and new insights. Biogerontology, 4(1): 1-8.

Bennett ST, Todd JA (1996) Human type diabetes and the insulin gene: principles of mapping polygenes. Annu. Rev. Genet., 30: 343-370.

Genotyping:

DNA was available for 1245 subjects of the combined cohorts of the Leiden 85-plus Study.

The genotypes for the five SNPs were determined with Taqman assays (Applied Biosystems, Nieuwerkerk, The Netherlands).

Assay by Design was used as recommended except for the following modifications:

A qPCR core kit was used (Eurogentec, Maastricht, The Netherlands) with one-third of the recommended amount of assay mix.

PCRs were performed on an ABI9700, and post-PCR fluorescence was measured on an ABI7900.

Reduced IIS and human longevity, D. van Heemst et al., growth hormone (GH) secretory bursts and the half-life of GH in healthy men. J. Clin. Endocrinol. Metab., 86(1): 292-299.

Kenyon C (2010) A conserved regulatory system for aging. Cell, 120(4): 437-448.

Kops GJ, Dansen TB, Polderman PE, Saarloos I, Wirtz KW, Coffer PJ, Huang TT, Bos JL, Medema RH, Burgering BM (2002) Forkhead transcription factor FOXO alpha protects quiescent cells from oxidative stress. Nature, 419(6904): 316-321.

Kurtzke JF (1983) On estimating survival; a tale of two censors. J. Clin. Epidemiol., 36(8): 771-778.

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