Candidate gene study of FOXO1, FOXO4, and FOXO6 reveals no association with human longevity in Germans. Rabea Kleindorp, Friederike Flachsbart, Annibale A. Puca, Alberto Malovini, Stefan Schreiber, and Almut Nebel conducted this study. The study was done at the Institute of Clinical Molecular Biology at Christian-Albrechts-University in Kiel, Germany; Multimedica in Milan, Italy; the Institute of Biomedical Technologies of the National Research Council in Segrate, Italy; the Department of Computer Engineering and Systems Science at the University of Pavia in Pavia, Italy; and the Biobank at Christian-Albrechts-University in Kiel, Germany.

Introduction

The forkhead box class O (FOXO) family of transcription factors is evolutionarily conserved and characterized by the so-called forkhead box DNA-binding domain. In mammals, the FOXO gene family consists of four members: FOXO1, FOXO3A, FOXO4, and FOXO6. These genes are homologous to the dauer formation-16 (daf-16) gene in the worm Caenorhabditis elegans (C. elegans). Numerous studies have shown that FOXO proteins play an important role in a wide range of normal biological processes, including cellular proliferation, cell cycle arrest, stress response, and apoptosis (Burgering & Kops, 2002; Stahl et al., 2002; Accili & Arden, 2004), as well as in diseases like cancer or diabetes mellitus (Furukawa-Hibi et al., 2002; Kato et al., 2006). Furthermore, FOXOs interact with several pathways that regulate cellular survival and aging. Early studies linking daf-16 in C. elegans as a key regulator of the insulin-IGF1 signaling (IIS) pathway (Kenyon, 2005) to extended lifespan (Tissenbaum & Guarente, 2001) demonstrated this.

Following these findings in model organisms, Willcox et al. (2008) recently identified one of the daf-16 homologues, FOXO3A, as a susceptibility gene for human longevity in ethnic Japanese from Hawaii. This association was subsequently replicated in German, Italian, Chinese, US-American, Jewish, and Danish populations. 

Summary

The forkhead box class O (FOXO) family of transcription factors consists of four members: FOXO1, FOXO3A, FOXO4, and FOXO6. The FOXO genes are homologues of daf-16, a key regulator of the insulin-IGF1 signaling pathway and a modulator of lifespan in Caenorhabditis elegans. Recently, variants in FOXO3A have consistently been associated with human longevity in various populations worldwide. Given this confirmed finding, it is conceivable that polymorphisms in the other FOXO genes might have a similar effect on human longevity.

To evaluate whether allelic variation in FOXO1, FOXO4, and FOXO6 influences the ability to become long-lived, we performed a comprehensive haplotype-tagging analysis of the three genes in a group of 1447 centenarians/nonagenarians and 1029 younger controls from Germany. This is the first investigation to analyze a possible association of human longevity with FOXO4 and FOXO6 and the largest and most comprehensive study to date to assess the genetic contribution of FOXO1 to the phenotype.

Our results suggest that none of the three genes plays a significant role in the ability to reach old age among Germans. With regard to FOXO1, this observation is supported by data from an Italian sample and is consistent with several previous reports but appears to be in contrast to a recent study of Han Chinese. The discordant association findings in Europeans and Chinese may be explained by their different FOXO1 linkage distributions.

Human longevity is considered a multi-factorial phenotype with a genetic contribution of about 25% (Christensen et al., 2006). The genetic component has been shown to become stronger with increasing age of the individuals (Hjelmborg et al., 2006; Gogele et al., 2010). Many genetic factors are likely to be involved, each having only a weak to moderate effect (Christensen et al., 2006). Up to date, the e4 allele of the apolipoprotein E (APOE) gene and variation in FOXO3A remain the only genetic determinants that have consistently and repeatedly been found to influence survival into old age in numerous populations worldwide (Christensen et al., 2006; Willcox et al., 2008; Anselmi et al., 2009; Flachsbart et al., 2009; Li et al., 2009; PawSuggesting a female-specific effect (Li et al., 2009), the results of the single-point analysis were replicated in an additional sample of Chinese. However, this finding contrasts with other studies that show no evidence of an association between FOXO1 and human longevity (Bonafe et al., 2003; Kojima et al., 2004; Willcox et al., 2008).

Results:
In this study, we analyzed 36 haplotype-tagging SNPs (htSNPs) in the gene regions of FOXO1, FOXO4, and FOXO6 for association with human longevity. The polymorphisms were spaced across the entire genes, covering most of the allelic variation. For none of the SNPs, we found a significant deviation from Hardy Weinberg equilibrium (HWE) in the control population (P > 0.02). Single-marker comparisons did not show a significant association for any of the tested SNPs, neither for the complete sample of LLI (Table 1) nor for the female (Table 2) or male subgroup (data not shown). A subsequent haplotype-based association analysis using a sliding window with pairs of consecutive SNPs revealed no association in any of the three genes with human longevity (data not shown).

Here, we performed a comprehensive analysis of FOXO1, FOXO4, and FOXO6 in a large group of 1447 long-lived individuals (LLI), i.e. centenarians and nonagenarians, and 1029 younger individuals.

Table 1: Association statistics for 36 haplotype-tagging single-nucleotide polymorphisms (htSNPs) in FOXO1, FOXO4, and FOXO6 in German LLI and controls.