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Keywords:

  • aging;
  • association study;
  • centenarians;
  • FOXO3A;
  • long-lived individuals

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Funding
  9. Author contributions
  10. References

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 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, 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 ability to reach old age. 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 discrepant association findings in Europeans and Chinese may be explained by their different FOXO1 linkage disequilibrium structures and could indicate a Chinese- or Asian-specific effect.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Funding
  9. Author contributions
  10. References

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 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, 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).

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; Gögele 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 ε4 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; Pawlikowska et al., 2009; Soerensen et al., 2010).

Given the confirmed association of FOXO3A variation with human longevity, it is conceivable that single-nucleotide 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 prospective population-based Leiden 85+ study revealed for carriers of a particular 3-SNP haplotype a 1.14-fold increased all-cause mortality risk (P value = 0.021, without correction for multiple testing) (Kuningas et al., 2007). More recently, statistically significant frequency differences for two SNPs and two haplotypes 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 replicated in an additional sample of Chinese. However, this finding is in contrast to other studies showing no evidence for an association of FOXO1 with human longevity (Bonafe et al., 2003; Kojima et al., 2004; Willcox et al., 2008).

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

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Funding
  9. Author contributions
  10. References

In this study, 36 haplotype-tagging SNPs (htSNPs) in the gene regions of FOXO1, FOXO4, and FOXO6 were analyzed for association with human longevity. The polymorphisms were spaced across the entire genes, covering the majority of the allelic variation. For none of the SNPs, a significant deviation from Hardy–Weinberg equilibrium (HWE) was found in the control population (> 0.02). Single-marker comparisons did not show a significant association in any of the tested SNPs, neither for the complete sample of LLI (Table 1) nor 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).

Table 1.   Association statistics for 36 haplotype-tagging single-nucleotide polymorphisms (htSNPs) in FOXO1, FOXO4, and FOXO6 in German LLI and controls
GeneChr.SNPbpminAF LIminAF controlsP alleleP genoOR (95% C.I.)
  1. Chr., chromosome; bp, base-position of NCBI build 36; minAF, minor allele frequency; LLI, long-lived individuals; P allele, P value obtained from allele-based case–control comparison using a chi square-test with 1 degree of freedom (df); P geno, P value obtained from genotype-based case–control comparison using a chi square-test with 2 df; OR, estimated odds ratio for minor allele (based on whole sample); CI, 95% confidence interval of OR; NA, genotype-based case–control comparison was not conducted when there were fewer than five observations. Associated markers from the study of Li et al. (2009) are in bold.

FOXO113rs2701893400236360.3490.3530.7720.5200.98 (0.87–1.11)
FOXO113rs2721047400256820.0860.0970.1770.2460.87 (0.72–1.06)
FOXO113rs17446593400260850.1700.1810.3380.6310.93 (0.80–1.08)
FOXO113rs2755209400358040.3730.3770.7700.2740.98 (0.871.11)
FOXO113rs1078892400360200.1870.1930.5890.4250.96 (0.83–1.11)
FOXO113rs2701859400392320.2520.2720.1170.0590.90 (0.79–1.03)
FOXO113rs12865518400411900.2500.2370.3070.3451.07 (0.94–1.23)
FOXO113rs2721069400417200.3100.3250.2510.1870.93 (0.82–1.05)
FOXO113rs2755213400443010.0900.0940.6260.8760.95 (0.781.16)
FOXO113rs2701880400485050.0600.0710.120NA0.83 (0.66–1.05)
FOXO113rs2951787400597700.3840.3760.6000.8341.03 (0.92–1.16)
FOXO113rs2984121400599790.1850.1780.5560.8231.05 (0.90–1.21)
FOXO113rs2721044400602250.3070.3300.0970.0740.90 (0.80–1.02)
FOXO113rs4943794400714080.2110.2230.3130.4570.93 (0.81–1.07)
FOXO113rs1986649400768240.2110.2250.2290.4340.92 (0.80–1.06)
FOXO113rs17630266400920320.0360.0330.510NA1.11 (0.81–1.52)
FOXO113rs12876443400948770.1070.1070.9890.8711.00 (0.83–1.20)
FOXO113rs7981045401072360.2730.2610.3490.5501.06 (0.93–1.21)
FOXO113rs9603776401218860.0250.0240.778NA1.05 (0.73–1.52)
FOXO4Xrs12013673702338240.4370.4430.6960.5480.98 (0.86–1.10)
FOXO4Xrs5981072702362670.2990.3110.3680.9760.94 (0.83–1.07)
FOXO61rs11585393416028440.3010.2930.5310.4661.04 (0.92–1.18)
FOXO61rs7539614416036340.4440.4390.7390.9041.02 (0.91–1.15)
FOXO61rs7547654416039480.4580.4400.2000.3561.08 (0.96–1.21)
FOXO61rs11581271416076480.4010.4230.1310.0970.91 (0.81–1.03)
FOXO61rs1317558416116700.1190.1080.2570.1951.11 (0.93–1.33)
FOXO61rs1317557416119290.0280.0270.824NA1.04 (0.73–1.48)
FOXO61rs6693260416120820.1750.1780.8110.8240.98 (0.85–1.14)
FOXO61rs4660532416127840.3310.3480.2240.4610.93 (0.82–1.05)
FOXO61rs6690527416145730.2890.2870.8670.3661.01 (0.89–1.15)
FOXO61rs4660192416149370.4580.4640.6630.8330.97 (0.87–1.09)
FOXO61rs11209971416168800.2810.2930.3360.5110.94 (0.83–1.07)
Table 2.   Association statistics for 36 haplotype-tagging single-nucleotide polymorphisms (htSNPs) in FOXO1, FOXO4, and FOXO6 in German female LLI and controls
GeneChr.SNPbpminAF LLIminAF controlsP alleleP genoOR (95% C.I.)
  1. Chr., chromosome; bp, base-position of NCBI build 36; minAF, minor allele frequency; LLI, long-lived individuals; P allele, P value obtained from allele-based case–control comparison using a chi square-test with 1 degree of freedom (df); P geno, P value obtained from genotype-based case–control comparison using a chi square-test with 2 df; OR, estimated odds ratio for minor allele (based on whole sample); CI, 95% confidence interval of OR; NA, genotype-based case–controls comparison was not conducted when there were fewer than five observations. Associated markers from the study of Li et al. (2009) are in bold.

FOXO113rs270189340 023 6360.3450.3530.6170.3480.97 (0.84–1.11)
FOXO113rs272104740 025 6820.0820.0980.091NA0.82 (0.66–1.03)
FOXO113rs1744659340 026 0850.1670.1820.2390.3830.90 (0.76–1.07)
FOXO113rs275520940 035 8040.3690.3760.6540.1090.97 (0.851.11)
FOXO113rs107889240 036 0200.1830.1960.2880.0540.92 (0.78–1.08)
FOXO113rs270185940 039 2320.2470.2690.1300.0780.89 (0.77–1.03)
FOXO113rs1286551840 041 1900.2560.2430.3830.5811.07 (0.92–1.24)
FOXO113rs272106940 041 7200.3090.3230.3670.3030.94 (0.82–1.08)
FOXO113rs275521340 044 3010.0870.0930.5480.8080.93 (0.751.17)
FOXO113rs270188040 048 5050.0580.0700.121NA0.81 (0.62–1.06)
FOXO113rs295178740 059 7700.3820.3720.5130.8061.05 (0.92–1.20)
FOXO113rs298412140 059 9790.1850.1810.8000.8981.02 (0.86–1.21)
FOXO113rs272104440 060 2250.3070.3280.1700.1350.91 (0.79–1.04)
FOXO113rs494379440 071 4080.2120.2250.3460.4020.93 (0.79–1.08)
FOXO113rs198664940 076 8240.2120.2270.2770.4130.92 (0.79–1.07)
FOXO113rs1763026640 092 0320.0360.0300.315NA1.20 (0.84–1.73)
FOXO113rs1287644340 094 8770.1090.1080.8800.9011.02 (0.82–1.25)
FOXO113rs798104540 107 2360.2780.2660.4350.6961.06 (0.92–1.23)
FOXO113rs960377640 121 8860.0270.0240.530NA1.14 (0.76–1.72)
FOXO4Xrs1201367370 233 8240.4320.4280.8000.5741.02 (0.89–1.16)
FOXO4Xrs598107270 236 2670.2980.2960.9270.9821.01 (0.87–1.16)
FOXO61rs1158539341 602 8440.2960.2880.6060.5371.04 (0.90–1.20)
FOXO61rs753961441 603 6340.4430.4370.7070.9241.03 (0.90–1.17)
FOXO61rs754765441 603 9480.4600.4490.4760.7631.05 (0.92–1.19)
FOXO61rs1158127141 607 6480.3950.4080.4460.4450.95 (0.83–1.09)
FOXO61rs131755841 611 6700.1210.1030.0840.0701.20 (0.98–1.47)
FOXO61rs131755741 611 9290.0280.0250.541NA1.14 (0.76–1.71)
FOXO61rs669326041 612 0820.1680.1790.3880.5910.93 (0.78–1.10)
FOXO61rs466053241 612 7840.3420.3580.3330.5980.94 (0.82–1.07)
FOXO61rs669052741 614 5730.2860.2830.8300.4051.02 (0.88–1.17)
FOXO61rs466019241 614 9370.4540.4480.7250.9391.02 (0.90–1.17)
FOXO61rs1120997141 616 8800.2810.2790.8870.9901.01 (0.87–1.17)

The markers used for the FOXO1 haplotype analysis in the Leiden 85+ study (Kuningas et al., 2007) were also subjected to a haplotype analysis in our sample (rs2721069, rs4943794, and rs7981045; Fig. 1A). The frequency of the observed mortality haplotype in the Dutch sample (TCA) was not significantly different in German LLI compared with younger controls (Fig. 1B).

image

Figure 1.  (A) Gene structure of FOXO1. (B) Results of the haplotype analysis in German long-lived individuals and controls. Single-nucleotide polymorphisms are based on the study of Kuningas et al. (2007). minAF, minor allele frequency.

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Recently, association signals for the FOXO1 SNPs rs2755209 and rs2755213 were reported in two samples of female Han Chinese (Li et al., 2009). In Germans, the frequencies of the polymorphisms did not differ significantly between the LLI and the young controls, neither for the complete sample (Table 1) nor the female subgroup (Table 2). When we investigated an additional population of female Italians for the same SNPs, this analysis did neither show evidence of association (Table 3). In view of the very similar FOXO1 SNP frequencies and linkage disequilibrium (LD) structures in Germans and Italians (Table 5 and Fig. 2), the data from the two populations were pooled. Also the combined sample with an a priori power of 84% did not confirm the previous finding of Li et al. (Table 3). Furthermore, in the German sample of female individuals (Table 4), we did not detect any association between the two haplotypes (ATG and CCG based on rs2755209, rs2755213, and rs17630266) and the longevity phenotype, as earlier reported in Chinese (Li et al., 2009).

Table 3.   Association statistics of FOXO1 single-nucleotide polymorphisms (SNPs) in female Italian individuals and in the pooled sample of Germans and Italians
  minAF LLIminAF controlsP alleleP genoOR (95% C.I.)
  1. SICS, South Italian Centenarian Study; minAF, minor allele frequency; LLI, long-lived individuals; n, number of individuals; P allele, P value obtained from allele-based case–control comparison using a chi square-test with 1 degree of freedom (df); P geno, P value obtained from genotype-based case–control comparison using a chi square-test with 2 df; OR, estimated odds ratio for minor allele (based on whole sample); CI, 95% confidence interval of OR; NA, genotype-based case–control comparison was not conducted when there were fewer than five observations.

SICS
 LLI = 166rs27552090.4040.3910.7280.3791.05 (0.79–1.41)
 Controls = 216rs27552130.1240.1180.795NA1.06 (0.68–1.64)
Germans + SICS
 LLI = 1258rs27552090.3730.3790.7020.6010.98 (0.86–1.10)
 Controls = 1010rs27552130.0920.0990.4450.2710.92 (0.76–1.13)
Table 5.   Comparison of minor allele frequencies of FOXO1 single-nucleotide polymorphisms (SNPs) in German, Italian, Chinese, and Japanese controls
 minAF controls GermansminAF controls ItaliansminAF controls Chinese†minAF controls Japanese‡
  1. Associated SNPs from Li et al. (2009) are in bold.

  2. minAF, minor allele frequency; NA, no information available.

  3. †Data from Li et al. (2009).

  4. ‡Data from Kojima et al. (2004).

  5. §Based on SNP rs2297627 from Kojima et al. (2004) that is in strong linkage disequilibrium (LD) with rs2755209 (r2 = 0.90, using JPT HapMap data).

  6. ¶Based on imputed data.

  7. ††Based on SNP rs2297626 from Kojima et al. (2004) that is in strong LD with rs17630266 (r2 = 0.95, using JPT HapMap data).

rs27552090.370.400.290.33§
rs27552130.090.110.42NA
rs176302660.030.02¶0.370.39††
image

Figure 2.  LD structure of FOXO1 in Germans (A), Italians (B), Chinese (C), and Japanese (D). The depicted single-nucleotide polymorphisms were used in the study of Chinese (Li et al., 2009). The linkage disequilibrium plots are based on the measure r2 and were generated with Haploview using HapMap data of Han Chinese (CHB) and Japanese (JPT) as well as data of German and Italian control individuals from this study.

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Table 4.   Comparison of FOXO1 haplotype association results in German and Chinese females
Haplotype†GermansChinese‡
Freq. casesFreq. controlsP valueFreq. casesFreq. controlsP value
  1. Associated haplotypes from Li et al. (2009) are in bold.

  2. freq., frequency.

  3. †Haplotype defined by the SNPs rs2755209, rs2755213, and rs17630266.

  4. ‡Data from Li et al. (2009).

  5. §Haplotype ATG was designated TTG in the study of Li et al. (2009).

ATG§0.6080.6140.7680.2300.2920.003
CTG0.2920.2870.8110.1160.1270.550
CCG0.0600.0610.9010.2480.1920.006
CCT0.0310.0270.5810.3920.3800.604
CTT0.0050.0070.4780.0080.0040.380

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Funding
  9. Author contributions
  10. References

In the present study, we performed comprehensive haplotype tagging of the FOXO gene family members FOXO1, FOXO4, and FOXO6 in an extensive collection of LLI and younger controls, comprising altogether 2476 German individuals. This is the first investigation to analyze a possible association between FOXO4 or FOXO6, respectively, and human longevity, and the largest and most comprehensive one to date to assess the genetic contribution of FOXO1 to the phenotype (1886 German and 382 Italian females). Our overall result suggests that polymorphisms in none of the genes have a significant influence on a person’s ability to reach old age.

For FOXO1, several longevity association studies have previously been performed, albeit with contradictory findings (Bonafe et al., 2003; Kojima et al., 2004; Li et al., 2009). Bonafe et al. (2003) described a negative result in Italians that is – though only a single SNP was tested – consistent with ours. Recently, a study of Han Chinese found two SNPs as well as two haplotypes in this gene to be associated with longevity in women (Li et al., 2009). The results of the single-point analysis were also replicated in an additional sample of Chinese (Li et al., 2009). However, two previous investigations of Asian populations had revealed no association (Kojima et al., 2004; Willcox et al., 2008). One of the studies that examined only male individuals of Japanese descent (Willcox et al., 2008) is not informative with regard to a potential association in females. Another report analyzing Japanese of both genders tested three SNPs (Kojima et al., 2004), which were not haplotype-tagging and thus did not cover the allelic variation in FOXO1 comprehensively. Of note, one of the SNPs, rs2297627, is in strong LD with one of the significantly associated markers described in Han Chinese (rs2755209, Table 5). A comparison of the two SNPs reveals a comparable increase in their minor allele frequencies in controls relative to LLI (2.9% in Japanese (Kojima et al., 2004) and 4.4% in Chinese (Li et al., 2009). In Japanese, this difference is not significant, which may be because of the relative small sample size used in that study (122 centenarians and 122 controls). Also the other two FOXO1 SNPs of Li et al. (2009) show similar allele frequencies in both Chinese and Japanese (Table 5), which raises the intriguing possibility of a continent-specific effect. This observation is underscored by the notable differences in LD structure and allele/haplotype distribution between European and Asian populations [Tables 4 and 5, Fig. 2 and HapMap data (Consortium 2003)]. Therefore, although our study did not show any evidence for an association of FOXO1 variants with human longevity in Germans and Italians, this does not exclude an effect that could be restricted to Chinese or Asians. Longevity in different populations is likely to be influenced by varying sets of interacting genetic and environmental factors (e.g. diet) (Caliebe et al., 2010). To confirm the Asian- and female-specific longevity association of FOXO1, replications with more extensive DNA collections of, for instance, Japanese origin would be helpful.

Polymorphisms in FOXO3A have convincingly and repeatedly been associated in numerous populations with the ability to survive into old age (Willcox et al., 2008; Anselmi et al., 2009; Flachsbart et al., 2009; Li et al., 2009; Pawlikowska et al., 2009; Soerensen et al., 2010). Although here we used a larger sample than the one genotyped for the FOXO3A analysis (Flachsbart et al., 2009), our results did not ascertain an association of FOXO1, FOXO4, or FOXO6 with longevity in Germans. The members of the FOXO gene family share some characteristics (more than 60% sequence identity of the human FOXO domain (Anderson et al., 1998), regulation via the IIS pathway and a seemingly functional redundancy in vitro (Coffer & Burgering, 2004)) and yet their physiological roles are thought to be diverse (Wang et al., 2009), a finding that is supported by different expression patterns (Furuyama et al., 2000; Biggs et al., 2001). This functional diversification is also illustrated by the disruption of the FOXO genes (Hosaka et al., 2004): while a homozygous knock-out of FOXO1 is embryonically lethal, FOXO3A−/− and FOXO4−/− animals are viable and initially develop normal. Further studies are needed to clarify in more detail the in vivo roles of the FOXO genes, and in particular of FOXO3A, in human longevity.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Funding
  9. Author contributions
  10. References

Study participants

The study sample comprised 1447 LLI with an age range of 95–110 years (mean age: 98.8 years) and 1029 younger controls (60–75 years; mean age: 66.8 years). About 75% of the LLI were women. The controls were similar to the LLI in terms of gender (75% women), ancestry, and geographic origin. There are no mortality data available for the controls. However, based on current predictions, only 1.5% of all 60-year-old and 1.8% of all 75-year-old German women will become 100 years. The study participants provided, as part of a health and family history questionnaire, personal and medical information. A detailed description of the study population and recruitment procedures is provided elsewhere (Nebel et al., 2005). All subjects gave written, informed consent prior to enrolment. The study was approved by the Ethics Committee of the University Hospital Schleswig-Holstein in Kiel.

The Italian women sample consisted of 166 LLI (90–109 years, mean age: 98.39 years) and 216 younger controls (18–48 years; mean age: 31.06 years) obtained from the Southern Italian Centenarian Study (SICS). SICS LLI were thoroughly investigated for demographic and clinical characteristics and they were enrolled by Associazione Longevità (Anselmi et al., 2009). All subjects gave written informed consent to the study. The study was approved by the Institutional Review Board of the ‘Istituto di Ricovero e Cura a Carattere Scientifico’, Multimedica, Milano, Italy.

Genotyping

Genotyping of the German samples was performed using the SNPlex™ Genotyping System and TaqMan® SNP Genotyping Assays (both Applied Biosystems, Foster City, CA, USA) on an automated platform (Hampe et al., 2001). For quality control purposes, positive controls on each genotyping plate were checked for consistency. All SNPs were tested for deviation from the HWE. The call rate for each marker exceeded 95.8%.

Genotyping of SICS individuals was carried out with the Infinium II Assay-HumanHap 317K duo BeadChip system (Illumina, San Diego, CA, USA) using standard protocols. Individual samples and markers showing a genotyping rate lower than 93% and 95%, respectively, were excluded. Imputation of unobserved genotypes was performed using Impute, version 0.5 (Marchini et al., 2007) with the HapMap CEU release 24 reference panel (http://www.hapmap.org/).

Statistical analysis

Haplotype-tagging SNPs (htSNPs) of FOXO1, FOXO4, and FOXO6 were selected based on the HapMap genotypes of Utah residents with ancestry from northern and western Europe (CEU; http://www.hapmap.org/) and the pairwise tagging option implemented in the Haploview v3.32 program (Barrett et al., 2005) (http://www.broad.mit.edu/mpg/haploview/) (minor allele frequency: > 0.05, pairwise r2 ≥ 0.8, PHWE: > 0.01, for htSNPs see Table 1). The exact genomic regions for the selection of the htSNPs were as follows: for FOXO1 chromosome 13: 40 022 274–40 144 279; for FOXO4 X chromosome: 70 227 715–70 245 118 and for FOXO6 chromosome 1: 41 596 756–41 624 373 (all NCBI36). The Haploview program was also applied for the haplotype analysis of FOXO1. An additional haplotype analysis using a two-marker sliding window was performed for all three genes with the sliding window option implemented in the programme COCAPHASE, version 2.403 that is part of PHASE, version 2.1 (http://www.stat.washington.edu/stephens/; Dudbridge, 2003). Single-point analysis was performed using PLINK v1.07 (Purcell et al., 2007). P < 0.05 was considered statistically significant. The a priori power calculation for rs2755209 in the German and Italian women sample was carried out with the PS Power and Sample Size Program (PS version 3.0.14; http://medipe.psu.ac.th/episoft/pssamplesize), assuming an OR of 0.77 as observed in the Chinese study (Li et al., 2009) and the minor allele frequency (40%) characteristic of the CEU HapMap sample.

Acknowledgments

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Funding
  9. Author contributions
  10. References

We thank all study participants for their cooperation and the laboratory personnel at the Institute of Clinical Molecular Biology and the Popgen biobank for excellent technical assistance.

Funding

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Funding
  9. Author contributions
  10. References

This study was supported by the DFG Excellence Cluster ‘Inflammation at Interfaces’.

Author contributions

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Funding
  9. Author contributions
  10. References

R.K., A.N., S.S. designed research; R.K. performed research; R.K., F.F., A.A.P, A.M. analyzed data; R.K, A.N. wrote the paper.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Funding
  9. Author contributions
  10. References
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