When comparing genotype frequencies, all SNPs except rs10499051, rs12212067, and rs12207868 were found to be nominally significantly associated with longevity (rs3800232 was significant only for the centenarians), see Supporting infor- mation Tables S2 and Table 1. Nine of these 12 SNPs (excluding rs12206094, rs13220810, and rs3800232), or SNPs reported to be in pronounced LD at these SNPs, have previously been reported to be associated at the genotype level in at least one study (Anselmi et al. 2009; Flachsbart et al. 2009; Pawlikowska et al. 2009; Willcox et al. 2008).

After correction for multiple testing (see Supporting informa- tion Table S3 and Table 2), 8 of the 12 SNPs remained signifi- cant; two for both genders combined (rs479744 and rs7762395) and eight when analyzing males separately (rs12206094, rs13220810, rs13217795, rs2764264, rs479744, rs7762395, rs9486902, and rs9400239). Four of them

Finally, studies published so far on the association of FOXO3A SNPs with longevity have been case–control carried out by comparing the group of oldest old to a younger control group. To further investigate the association of FOXO3A SNPs with extreme survival, we investigated the 15 SNPs using longi-

tudinal survival data for our homogenous cohort of oldest old. If fulfilling the proportional hazard assumptions, the Cox pro- portional hazard model was applied. Three SNPs were found to be nominally associated with longevity: rs10499051 in females and in both genders combined and rs7762395 and rs9486902 in males.

cation, however, because of minimal immigration into both these cohorts, the cohorts must be considered to be genetically

In the case–control part of the study presented here, we inves- tigate the association of FOXO3A SNPs in the controls when compared to the entire group of oldest old (N = 1089), as well as of those individuals from the 1905 cohort who became cente- narians (N = 143). The reason for doing so is that the association of FOXO3A SNPs with longevity has previously been reported to be most pronounced for centenarians (Flachsbart et al. 2009).

rs10499051 was not found to be significant in the case-con- trol study. For rs7762395 and rs9486902, an increase in the rare genotype from middle age to old age was observed in the case– control study, which seems contradictory to the survival disad- vantage during old age. The latter might, however, be explained by antagonistic pleiotropy, i.e., the two SNPs might have a posi- tive effect on survival from middle age until old age, yet a nega- tive effect during old age. However, none of these three SNPs hold significance after correction for multiple testing (via Bonfer- roni); hence, it might simply be chance findings and, hence, we cannot conclude of an association based on the longitudinal

Both surveys included multidimensional face-to-face inter- views, assessment of functional and cognitive abilities and DNA sampling. Permission to collect blood samples and usage of reg- ister-based information was granted by The Danish National Committee on Biomedical Research Ethics.

Selection of chromosome region and tagging SNPs

In this study, we replicated some of the previous findings regarding an association of variation in the FOXO3A gene with human longevity using a case–control approach and, moreover, found novel SNPs to be associated. In addition, we report of a longitudinal analysis, which did, however, not show results hold- ing for the correction of multiple testing. Hence, our findings add further evidence to the role of FOXO3A as a longevity candi- date gene from younger ages to old age, yet not during old age.

The genomic region investigated in this study is position 108,982,719–109,113,664 of chromosome 6, which corre- sponds to FOXO3A and 5000 base pair (bp) upstream and 1000 bp down stream (NCBI assemble 36). Data about variation in this gene region were obtained through the HapMap consor- tium database (http://hapmap.ncbi.nlm.nih.gov/index.html.en) for the CEPH (Utah residents with ancestry from northern and western Europe (CEPH) (CEU)) cohort, using the HapMap Data Rel 23a⁄phase II Mar08, on NCBI B36 assembly, dbSNP b 126 criteria. Genotype SNP data were analyzed using the HaploView software (http://www.broadinstitute.org/haploview/haploview, Barrett et al. (2005)) to choose tagging SNPs covering 100% of the known common genetic variation in FOXO3A (‘pair wise tagging only’, R2 = 0.8, LOD = 3 and a minimum distance between SNPs = 60 bp criteria were used). All tagging and can- didate SNPs included in this study have a minor allele frequency (MAF) of at least 5%.

Experimental procedures

Subjects

The oldest old individuals of our study (N = 1089) were pants in The Danish 1905 Cohort Study, which includes Danes born in 1905 (Nybo et al. 2001). The cohort were assessed for the first time in 1998, when they 92–93 years of age. Survivors were subsequently assessed second year through 2005, and vital status followed until ary 1st 2010 or until death, whichever came first, resulting in mean follow-up time for the survivors of 11.4 years 11.2–11.6). Information on survival status was retrieved the Danish Central Population Register, which is updated (Pedersen et al. 2006). The younger control (N = 736) of this study was randomly selected from the Study Middle-Aged Danish Twins (Skytthe et al. 2002), which was tiated in 1998, when 2640 intact twin pairs from 22 birth years (1931–1952) were randomly selected via the Central Person Registry. The participants have been longitudinally through different registers (including Denmark). The control group in the study presented includes only one twin from each twin pair. Of the 736 als, 35 had died since the beginning of the survey in 1998, only approximately 9% of the 736 individuals are expected turn 93 years of age (this estimate is based on period life data for the Danish population (http://www.mortality.org)). potential confounder in association studies is population