Candidate gene study of FOXO1, FOXO4, and FOXO6 reveals no association with human longevity in Germans

Rabea Kleindorp,1 Friederike Flachsbart,1 Annibale A. Puca,2,3 Alberto Malovini,4 Stefan Schreiber1,5 and Almut Nebel1

of Clinical Molecular Biology, Christian

Albrechts

of Kiel, Kiel, Germany Multimedica, Milan, Italy di Tecnologie Biomediche – Consiglio Nazionale delle Segrate, Italy of Computer Engineering and Systems Science, of Pavia, Pavia, Italy Biobank, Christian

Albrechts

University of Kiel, Kiel,

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, which are homologous to the dauer for- mation-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 pro- cesses, 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 regu- late cellular survival and aging, as it was demonstrated by 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). Following on from these findings in model organisms, Willcox et al. (2008) have recently identified one of the daf-16 homologues, FOXO3A, as a susceptibility gene for human longevity in ethnic Japanese from Hawaii (Willcox et al., 2008). This association was subsequently replicated in German, Italian, Chinese, US-American, Jewish, and Danish populations (Anselmi et al., 2009; Flachsbart et al., 2009; Li et al., 2009; Pawlikowska et al., 2009; Soerensen et al., 2010).

Summary

In mammals, the forkhead box class O (FOXO) family of transcription factors consists of the four members FOXO1, FOXO3A, FOXO4, and FOXO6. The FOXO genes are homo- logues of daf-16, a key regulator of the insulin-IGF1 signaling pathway and a modulator of lifespan in Caenor- habditis 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 longev- ity. 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 centenari- ans⁄nonagenarians and 1029 younger controls from Ger- many. This is the first investigation to analyze a possible association of human longevity with FOXO4 and FOXO6, respectively, and the largest and most comprehensive study to date to assess the genetic contribution of FOXO1 to the phenotype. Our results suggest that in Germans, none of the three genes plays a significant role in the abil- ity to reach old age. With regard to FOXO1, this observa- tion 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 dis- crepant association findings in Europeans and Chinese may be explained by their different FOXO1 linkage dis-

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; Go¨gele et al., 2010). Many genetic factors are likely to be involved, each having only a weak to moderate effect (Christen- sen 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; Ansel- mi et al., 2009; Flachsbart et al., 2009; Li et al., 2009; Paw- likowska et al., 2009; Soerensen et al., 2010).

Almut Nebel, Institute of Clinical Molecular Biology, Christian-Albrechts-Uni- versity, Schittenhelmstrasse 12, 24105 Kiel, Germany. Tel.: +49 431 597 1373; fax: +49 431 597 2196; e-mail: a.nebel@mucosa.de

Given the confirmed association of FOXO3A variation with human longevity, it is conceivable that single-nucleotide

Accepted for publication 16 February

controls from Germany to evaluate whether allelic variation in any of the three genes influences the ability to attain old age.

polymorphisms (SNPs) in the other FOXO members, which are also regulated by the IIS pathway, might have a similar effect on the phenotype. Previously, an analysis of FOXO1 in the prospec- tive population-based Leiden 85+ study revealed for carriers of a particular 3-SNP haplotype a 1.14-fold increased all-cause mor- tality risk (P value = 0.021, without correction for multiple test- ing) (Kuningas et al., 2007). More recently, statistically significant frequency differences for two SNPs and two haplo- types in FOXO1 were reported between long-lived and younger women from China, suggesting a female-specific effect (Li et al., 2009). The results of the single-point analysis were repli- cated in an additional sample of Chinese. However, this finding is in contrast to other studies showing no evidence for an associ- ation of FOXO1 with human longevity (Bonafe et al., 2003; Koj- ima et al., 2004; Willcox et al., 2008).

Results