Replication of an association of variation in the FOXO3A gene with human longevity using both case-control and longitudinal data. Mette Sorensen, Serena Dato, Kaare Christensen, Matt McGue, Tinna Stevnsner, Vilhelm A. Bohr, and Lene Christiansen. The Danish Aging Research Center, Epidemiology, Institute of Public Health, University of Southern Denmark, Odense C, Denmark; Department of Clinical Genetics, Odense University Hospital, Odense C, Denmark; Department of Cell Biology, University of Calabria, Rende (Cs), Italy; Department of Psychology, University of Minnesota, MN, USA; The Danish Aging Research Center, Department of Molecular Biology, University of Aarhus, Aarhus C, Denmark; Laboratory of Molecular Gerontology, National Institute on Aging, National Institute of Health, Baltimore, MD, USA.

Introduction: Genetic factors contribute to the variation in human lifespan by approximately 25% (Herskind et al. 1996), a contribution believed to be minimal before age 60 years and most profound from age 85 years onwards (Hjelmborg et al. 2006). Candidate longevity genes encode proteins involved in several biological processes including the insulin-IGF-1 signaling pathway (Christensen et al. 2006), in which the transcription factor forkhead box O3A (Foxo3a) is of key importance.

Summary: Variation in the gene encoding Foxo3a (FOXO3A) has previously been reported to be associated with human longevity. Willcox et al. (2008) first reported on the associations of FOXO3A single nucleotide polymorphisms (SNPs) with longevity in male Americans of Japanese ancestry (rs2764264 (P-value (P) = 0.0002), rs13217795 (P = 0.0006) and rs2802292 (P < 0.0001)), associations which were subsequently confirmed (with effects pointing in the same direction) in Italian, American (northern and western European ancestry), Chinese, and German populations (Anselmi et al. 2009; Flachsbart et al. 2009; Li et al. 2009; Pawlikowska et al. 2009). These studies reported 6 of 16 tagging SNPs (five specific to males and one to females) (Anselmi et al. 2009), 3 of 16 tagging SNPs (both genders) (Flachsbart et al. 2009), 2 of 16 tagging SNPs (specific to females) (Pawlikowska et al. 2009), and 3 of 3 tagging SNPs (both genders) (Li et al. 2009) to be associated with longevity. Furthermore, two studies reported on the associations of FOXO3A haplotypes with longevity (Anselmi et al. 2009; Li et al. 2009).

Genetic variation in FOXO3A has previously been associated with human longevity. Studies published so far have been case-control studies and hence vulnerable to bias introduced by cohort effects. In this study, we extended the previous findings in the cohorts of oldest-old Danes (the Danish 1905 cohort, N = 1089) and middle-aged Danes (N = 736), applying a longitudinal study design as well as the case-control study design. Fifteen SNPs were chosen to cover the known common variation in FOXO3A. Comparing SNP frequencies in the oldest-old with middle-aged individuals, we found an association (after correction for multiple testing) of eight SNPs; four (rs13217795, rs2764264, rs479744, and rs9400239) previously reported to be associated with longevity and four novel SNPs (rs12206094, rs13220810, rs7762395, and rs9486902) (corrected P-values 0.001-0.044). Moreover, we found an association of the haplotypes TAC and CAC of rs9486902, rs10499051, and rs12206094 (corrected P-values: 0.01-0.03) with longevity. Finally, we present data applying a longitudinal study design; when using follow-up survival data on the oldest-old in a longitudinal analysis, we found no SNPs to remain significant after the correction for multiple testing (Bonferroni correction).

Studies comparing oldest-old to younger controls have raised concerns about possible cohort effects introducing bias (Beekman et al. 2006; Christensen et al. 2006). In this study, we aimed to add further support and to extend the evidence for a role of FOXO3A in longevity by investigating 15 FOXO3A SNPs covering the knownProcessed text:

The table includes P-values obtained from genotype-based case-control comparison. N represents the number of individuals in the group, OR is the odds ratio, and 95% CI is the 95% confidence interval. NA means not analyzed since no homozygotes (rare genotype) were found. The table assumes a recessive model and a dominant model. The data for males and females are separated, and the PCCG values are listed as uncorrected and corrected. 

To investigate the haplotype structure in more detail, the Plink software's sliding window feature was applied using a window of three SNPs. The haplotype frequencies and P-values from the haplotype frequency-based case-control comparison (PCCH) between middle-aged and oldest old are listed in Supporting Information Table S4, while data that are nominally significant and data that pass correction of multiple testing are shown in Table 3. When correcting for multiple testing, only one window (and two haplotypes) held for correction: window 1 (rs9486902, rs10499051, and rs12206094) haplotypes TAC and CAC were found to be significant for males only (see Table 3). For both genders combined, haplotypes in windows 1, 2, 3, and 4 were found to be nominally significant.

The case-control association analysis of FOXO3A haplotypes with longevity is divided into males and females combined (A) and for males separately (B). The table includes window SNPs in the window, haplotype, uncorrected PCCH, corrected PCCH columns. Only PCCHs being nominally significant (P < 0.05) are included in the table; data passing nominal significance are listed in columns named Uncorrected PCCH. Values passing correction by multiple testing are in bold. N represents the number of individuals in the group.