Association of FOXO3A variation with human longevity confirmed in German centenarians

Friederike Flachsbarta, Amke Caliebeb, Rabea Kleindorpa, He´le`ne Blanche´c, Huberta von Susanna Nikolausd, Stefan Schreibera,1,2, and Almut Nebela,1

aInstitute of Clinical Molecular Biology, bInstitute of Medical Informatics and Statistics, and dPopgen Biobank, Christian-Albrechts-University, 24105 Kiel, Germany; and cFondation Jean Dausset, Centre d’E´tude du Polymorphisme Humain, 75010 Paris, France

Edited by Cynthia J. Kenyon, University of California, San Francisco, CA, and approved December 31, 2008 (received for review September 25, 2008)

notypes in a discovery sample of long-lived Americans of Japanese ancestry. However, this finding required replication in an independent population. Here, we have investigated 16 known SNPs, which capture the majority of the variation in FOXO3A via its common haplotypes, in an extensive collection of 1,762 German centenarians, nonagenarians, and younger controls and provide evidence that polymorphisms in this gene are indeed associated with the ability to attain exceptional old age. Our findings confirmed the initial discovery in the Japanese sample and thus support FOXO3A as a susceptibility gene for prolonged survival in humans.

The human forkhead box O3A gene (FOXO3A) encodes an evolu- tionarily conserved key regulator of the insulin–IGF1 signaling pathway that is known to influence metabolism and lifespan in model organisms. A recent study described 3 SNPs in the FOXO3A gene that were statistically significantly associated with longevity in a discovery sample of long-lived men of Japanese ancestry [Willcox et al. (2008) Proc Natl Acad Sci USA 105:13987–13992]. However, this finding required replication in an independent population. Here, we have investigated 16 known FOXO3A SNPs in an extensive collection of 1,762 German centenarians/nonagenar- ians and younger controls and provide evidence that polymor- phisms in this gene were indeed associated with the ability to attain exceptional old age. The FOXO3A association was consid- erably stronger in centenarians than in nonagenarians, highlight- ing the importance of centenarians for genetic longevity research. Our study extended the initial finding observed in Japanese men towomenandindicatesthatbothgenderswerelikelytobeequally affected by variation in FOXO3A. Replication in a French centenar- ian sample generated a trend that supported the previous results. Our findings confirmed the initial discovery in the Japanese sample and indicate FOXO3A as a susceptibility gene for prolonged sur- vival in humans.

Results

In the present study, 16 polymorphisms in FOXO3A were analyzed for association with the human longevity phenotype (Tables 1 and 2). The tested SNPs are spaced across the entire gene region, including the promoter (Fig. 1) and capture the majority of its allelic variation by haplotype tagging. All SNPs were in Hardy-Weinberg equilibrium (HWE) in the control population. For the association analyses, we applied an estab- lished longevity study design (13, 14) by comparing German   long-lived individuals (LLI; subset A; n 1,031; aged 95–110 yt years) and a centenarian subset (subset B; n 388) to appro- FO priately matched younger controls (n 731; aged 60–75 years). All markers were subjected to allelic case-control comparisons (CCA) by using the entire LLI sample (subset A) and the centenarian subset (subset B). For subset A, single-marker analysis revealed 4 SNPs with nominally significant P values CCA (Table 1). For the centenarians (subset B), 11 SNPs showed significant association (Table 2). Although subset B is smaller in size and therefore expected to have less power than the overall LLI sample, the significance level was more pronounced in the centenarians and revealed a stronger effect as reflected in the odds ratios (ORs) (Tables 1 and 2). The 3 top-ranking FOXO3A markers in subset B (rs3800231, rs9400239, and rs479744) passed correction for multiple testing (Bonferroni-adjusted significance   SEASx | threshold 0.0016; for 2 16 tests). Because this adjustment did not take into account the strong linkage disequilibrium (LD) between the investigated markers (Fig. 1), the obtained thresh- old must be regarded as conservative. The results from the comparison of the genotypic data (CCG) are presented as additional information but they were not included in the initial statistical assessment (Tables 1 and 2). Because the age of the study participants seemed to influence the strength of the

aging  forkhead box O3A . genetic association study / long-lived individuals

mental and genetic factors. Approximately 25–32% of the overall variation in adult lifespan is accounted for by genetic differences that become particularly important for survival after the age of 60 (1–5). The mechanisms influencing lifespan have been intensively studied in Caenorhabditis elegans, Saccharomy- ces cerevisiae, or Drosophila melanogaster, and hundreds of genetic variants that lead to life extension in model systems have been identified (6–8). The success in finding lifespan-control genes in lower organisms has also motivated efforts to search for corresponding genes in humans. However, to date variation in only 1 gene, which codes for apolipoprotein E (APOE), has been found to be consistently associated with survival in various populations. Although numerous case-control candidate studies have been performed and associations of the longevity pheno- type with biologically plausible genes have been described, results from these experiments have proven difficult to validate (5). These findings emphasize the importance of conducting large-scale studies with adequate replication to identify variants that are likely to exhibit only a weak or moderate effect.