Genotyping

DNA was isolated from blood spot samples using the QIAamp DNA Mini and Micro Kits (Qiagen, Dusseldorf, Germany). Geno- typing of the 15 FOXO3A SNPs was performed using the Illumina GoldenGateplatform(IlluminaInc,SanDiego,CA,USA)anddata cleanup was performed according to Illumina Inc’s recommenda- tions (http://www.illumina.com/documents/products/technotes/ technote_infinium_genotyping_data_analysis.pdf). Finally, 24 and 48 DNA samples were included twice in the GoldenGate assay to investigate the intra-plate and the inter-plate reproduc- ibility.Datashowedanintra-platereproducibilityof99.4%(using 24 samples) and an inter-plate reproducibility of 96.8% (using 48 samples).

Statistics

The Plink statistical program (http://pngu.mgh.harvard.edu/ purcell/plink, (Purcell et al. 2007)) was used for investigating

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genotype, allele, and haplotype frequencies in the different groups and for association studies using v2 test statistics. P-val- ues were obtained for allelic case–control comparison (PCCA), for genotypic case–control comparison (PCCG), and for haplo- typic case–control comparison (PCCH). Analysis of differences in genotype frequencies was carried out by assuming firstly a genotype (assumption free) model and secondly recessive and dominant models. Haplotypes were investigated using the ‘Slid- ing window’ application in Plink, by setting the window at three SNPs sliding along the 15 SNPs in the order defined by their posi- tions on chromosome 6.

The longitudinal survival data analysis on the oldest old was conducted by using the STATA 10.0 statistical program (Stata Corporation, College Station, TX, USA) for estimating the mortality risk using the Cox proportional hazard model.

A nominal significance level was set at P < 0.05, and the per- mutation application in Plink was used for correcting for multi- ple testing of the allele, genotype, and haplotype data (applying max(T) permutation mode set at 1000 permutations), while for the Cox regression analysis, a Bonferroni correction level at P = 0.05/2.15 SNPs = 0.00166 was used.

Acknowledgments

This study was financially supported by the Max-Planck Institute for Demographic Research, (Rostock, Germany), the National Institute on Aging (P01 AG08761), the INTERREG 4 A pro- gramme Syddanmark-Schleswig-K.E.R.N (by EU funds from the European Regional Development Fund), the Novo Nordisk Foun- dation, the Aase and Ejnar Danielsen Foundation, the Augusti- nus Foundation, the Brødrene Hartmann Foundation, the King Christian the 10th foundation, and the Einer Willumsens Mindelegat Foundation. The Danish Aging Research Center is supported by a grant from the VELUX Foundation. Susanne Knudsen, Steen Gregersen, Ulla Munk and Shuxia Li are thanked for excellent technical work.

Author contributions

M.S., S.D., K.C., M.M., L.C., T.S., V.A.B.: Generating the con- ception of the study and the study design; M.S.: Acquisition of data, conduction of data analysis, and interpretation of data. Drafting the manuscript; S.D.: Contribution to the acquisition of data; K.C., M.M: Interpretation of data. L.C.: Acquisition of data and interpretation of data. Authors have revised the manuscript and given their final approval.

Supporting information

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