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Forkhead box (FOX)O proteins are transcription factors (TFs) with four members in mammals designated FOXO1, FOXO3, FOXO4, and FOXO6. FOXO TFs play a pivotal role in the cellular adaptation to diverse stress conditions. FOXO proteins act as context-dependent tumor suppressors and their dysregulation has been implicated in several age-related diseases. FOXO3 has been established as a major gene for human longevity. Accordingly, FOXO proteins have emerged as potential targets for the therapeutic development of drugs and geroprotectors. In this review, we provide an overview of the most recent advances in our understanding of FOXO regulation and function in various pathological conditions. We discuss strategies targeting FOXOs directly or by the modulation of upstream regulators, shedding light on the most promising intervention points. We also reveal the most relevant clinical indications and discuss the potential, trends, and challenges of modulating FOXO activity for therapeutic purposes., This work was supported by the Spanish Ministry of Science, Innovation, and Universities through Grant RTI2018-094629-B-I00 to W.L. This work was supported by the Kuakini Medical Center, the US National Institutes of Health [contract N01-AG-4-2149, grants 5U01AG019349-05, 5R01AG027060 (Kuakini Hawaii Lifespan Study) (B.J.W.), 5R01AG038707 (Kuakini Hawaii Healthspan Study), and 1P20GM125526-01A1 (Kuakini Center of Biomedical Research Excellence for Clinical and Translational Research on Aging) (B.J.W.)], contract N01-HC-05102 from the National Heart, Lung, and Blood Institute (B.J.W.), P01CA214274 from the National Cancer Institute (J.P.), MCL-RI from the Leukemia and Lymphoma Society (J.P.), and R56AG048284 from the National Institute of Aging (J.P.). J.B. thanks the Fundação para a Ciência e Tecnologia (FCT) and the Comissão de Coordenação e Desenvolvimento Regional do Algarve (CCDR Algarve) for supporting the work with the project ALG-01-0145-FEDER-28044 ‘VITAL’, PTDC/BTM-TEC/28044/2017, and the project ALG-01-0145-FEDER-072586 ‘ALFA Score’ #072586. This review is supported by STRATAGEM COST Action, CA17104.

Since the G allele of forkhead box O3 ( FOXO3 ) single nucleotide polymorphism (SNP) rs2802292 is associated with resilience and longevity, ostensibly by mitigating the adverse effects of chronic cardiometabolic stress on mortality, our aim was to determine the association between the FOXO3 SNP rs2802292 genotype and risk of hypertension-mediated intracerebral haemorrhage (ICH).From a prospective population-based cohort of Japanese American men from the Kuakini Honolulu Heart Program (KHHP), age-adjusted prevalence of ICH by hypertension was assessed for the whole cohort after stratifying by FOXO3 genotype. Cox regression models, adjusted for age, cardiovascular risk factors and, FOXO3 and APOE genotypes, were utilized to determine relative risk of hypertension's effect on ICH. All models were created for the whole cohort and stratified by FOXO3 G -allele carriage vs. TT genotype.Among 6469 men free of baseline stroke, FOXO3 G -allele carriage was seen in 3009 (46.5%) participants. Overall, 183 participants developed ICH over the 34-year follow-up period. Age-adjusted ICH incidence was 0.90 vs. 1.32 per 1000 person-years follow-up in those without and with hypertension, respectively ( P = 0.002). After stratifying by FOXO3 genotype, this association was no longer significant in G allele carriers. In the whole cohort, hypertension was an independent predictor of ICH (relative risk [RR] = 1.70, 95% confidence interval [CI] 1.25, 2.32; P = 0.0007). In stratified analyses, hypertension remained an independent predictor of ICH among the FOXO3 TT -genotype group (RR = 2.02, 95% CI 1.33, 3.07; P = 0.001), but not in FOXO3 G -allele carriers (RR = 1.39, 95% CI 0.88, 2.19; P = 0.15).The longevity-associated FOXO3 G allele may attenuate the impact of hypertension on ICH risk.

FOXO3 is a ubiquitous transcription factor expressed in response to cellular stress caused by nutrient deprivation, inflammatory cytokines, reactive oxygen species, radiation, hypoxia, and other factors. We showed previously that the association of inherited FOXO3 variants with longevity was the result of partial protection against mortality risk posed by aging-related life-long stressors, particularly cardiometabolic disease. We then referred to the longevity-associated genotypes as conferring “mortality resilience.” Serum proteins whose levels change with aging and are associated with mortality risk may be considered as “stress proteins.” They may serve as indirect measures of life-long stress. Our aims were to (1) identify stress proteins that increase with aging and are associated with an increased risk of mortality, and (2) to determine if FOXO3 longevity/resilience genotype dampens the expected increase in mortality risk they pose. A total of 4500 serum protein aptamers were quantified using the Somalogic SomaScan proteomics platform in the current study of 975 men aged 71–83 years. Stress proteins associated with mortality were identified. We then used age-adjusted multivariable Cox models to investigate the interaction of stress protein with FOXO3 longevity-associated rs12212067 genotypes. For all the analyses, the p values were corrected for multiple comparisons by false discovery rate. This led to the identification of 44 stress proteins influencing the association of FOXO3 genotype with reduced mortality. Biological pathways were identified for these proteins. Our results suggest that the FOXO3 resilience genotype functions by reducing mortality in pathways related to innate immunity, bone morphogenetic protein signaling, leukocyte migration, and growth factor response.

Introduction: Genetic variation in the phosphatidylinositol 3-kinase reregulatory subunit 1 gene (PIK3R1) is associated with longevity. Objective: The aim of the study was to determine whether cardiovascular disease (CVD) affects this association. Methods: We performed a longitudinal study of longevity-associated PIK3R1 single-nucleotide polymorphism rs7709243 genotype by CVD status in 3,584 elderly American men of Japanese ancestry. Results: At baseline (1991–1993), 2,254 subjects had CVD and 1,314 did not. The follow-up until Dec 31, 2019 found that overall, men with a CVD had higher mortality than men without a CVD (p = 1.7 × 10−5). However, survival curves of CVD subjects differed according to PIK3R1 genotype. Those with longevity-associated PIK3R1 TT/CC had survival curves similar to those of subjects without a CVD (p = 0.11 for TT/CC, and p = 0.054 for TC), whereas survival curves for CVD subjects with the CT genotype were significantly attenuated compared with survival curves of subjects without a CVD (p = 0.0000012 compared with TT/CC, and p = 0.0000028 compared with TC). Men without CVD showed no association of longevity-associated genotype with life span (p = 0.58). Compared to subjects without any CVD, hazard ratios for mortality risk were 1.26 (95% CI, 1.14–1.39; p = 0.0000043) for CT subject with CVD and 1.07 (95% CI 0.99–1.17; p = 0.097) for CC/TT subjects with CVD. There was no genotypic effect on life span for 1,007 subjects with diabetes and 486 with cancer. Conclusion: Our study provides novel insights into the basis for PIK3R1 as a longevity gene. We suggest that the PIK3R1 longevity genotype attenuates mortality risk in at-risk individuals by protection against cellular stress caused by CVD.

The single nucleotide polymorphism (SNP) rs4130113 of the growth hormone receptor gene (GHR) is associated with longevity. Here we explored whether longevity-associated genotypes protect against mortality in all individuals, or only in individuals with aging-related diseases. Rs4130113 genotypes were tested for association with mortality in 3,557 elderly American men of Japanese ancestry. At baseline (1991–1993), 1,000 had diabetes, 730 had coronary heart disease (CHD), 1,901 had hypertension, 485 had cancer, and 919 lacked these diseases. The men were followed from baseline until Dec 31, 2019 or death (mean 10.8 ± 6.5 SD years, range 0.01–28.8 years; 99.0% deceased by that date). In a heterozygote disadvantage model, longevity-associated genotypes were associated with significantly lower mortality risk in individuals having hypertension (covariate-adjusted hazard ratio [HR] 0.83 [95% CI: 0.76–0.93, p = 4.3 x10–4]. But in individuals with diabetes, CHD, and cancer there was no genotypic difference in lifespan. As expected, normotensive men outlived men with hypertension (p = 0.036). There was no effect, however, of genotypic difference on lifespan in normotensive men (p = 0.11). We found that SNP rs4130113 potentially influenced the binding of transcription factors E2A, MYF, NRSF, TAL1, and TCF12 so as to alter GHR expression. We propose that in individuals with hypertension, longevity-associated genetic variation in GHR enhances cell resilience mechanisms to help protect against cellular stress caused by hypertension. As a result, hypertension-affected men who possess the longevity-associated genetic variant of GHR live as long as normotensive men.

Genetic variants of the kinase signaling gene MAP3K5 are associated with longevity. Here we explore whether the longevity-association involves protection against mortality in all individuals, or only in individuals with aging-related diseases. We tested the strongest longevity associated single nucleotide polymorphism (SNP), rs2076260, for association with mortality in 3,516 elderly American men of Japanese ancestry. At baseline (1991-1993), 2,461 had either diabetes (n=990), coronary heart disease (CHD; n=724), or hypertension (n=1,877), and 1,055 lacked any of these cardiometabolic diseases (CMDs). The men were followed from baseline until Dec 31, 2019. Longevity-associated genotype CC in a major allele homozygote model, and CC+TT in a heterozygote disadvantage model were associated with longer lifespan in individuals having a CMD (covariate-adjusted hazard ratio [HR] 1.23 [95% CI: 1.12-1.35, p=2.5x10-5] in major allele homozygote model, and 1.22 [95% CI: 1.11-1.33, p=1.10x10-5] in heterozygote disadvantage model). For diabetes, hypertension and CHD, HR p-values were 0.019, 0.00048, 0.093, and 0.0024, 0.00040, 0.0014, in each respective genetic model. As expected, men without a CMD outlived men with a CMD (p=1.9x10-6). There was, however, no difference in lifespan by genotype in men without a CMD (p=0.21 and 0.86, respectively, in each genetic model). In conclusion, we propose that in individuals with a cardiometabolic disease, longevity-associated genetic variation in MAP3K5 enhances resilience mechanisms in cells and tissues to help protect against cardiometabolic stress caused by CMDs. As a result, men with CMD having longevity genotype live as long as all men without a CMD.

Life expectancy has increased substantially over the last 150 years. Yet this means that now most people also spend a greater length of time suffering from various age-associated diseases. As such, delaying age-related functional decline and extending healthspan, the period of active older years free from disease and disability, is an overarching objective of current aging research. Geroprotectors, compounds that target pathways that causally influence aging, are increasingly recognized as a means to extend healthspan in the aging population. Meanwhile, FOXO3 has emerged as a geroprotective gene intricately involved in aging and healthspan. FOXO3 genetic variants are linked to human longevity, reduced disease risks, and even self-reported health. Therefore, identification of FOXO3-activating compounds represents one of the most direct candidate approaches to extending healthspan in aging humans. In this work, we review compounds that activate FOXO3, or influence healthspan or lifespan in a FOXO3-dependent manner. These compounds can be classified as pharmaceuticals, including PI3K/AKT inhibitors and AMPK activators, antidepressants and antipsychotics, muscle relaxants, and HDAC inhibitors, or as nutraceuticals, including primary metabolites involved in cell growth and sustenance, and secondary metabolites including extracts, polyphenols, terpenoids, and other purified natural compounds. The compounds documented here provide a basis and resource for further research and development, with the ultimate goal of promoting healthy longevity in humans.

FOXO3 is a prominent longevity gene. To date, no-one has examined whether longevity-associated FOXO3 genetic variants protect against mortality in all individuals, or only in those with aging-related diseases. We therefore tested longevity-associated FOXO3 single nucleotide polymorphisms in a haplotype block for association with mortality in 3,584 elderly American men of Japanese ancestry, 2,512 with and 1,072 without a cardiometabolic disease (CMD). At baseline (1991–1993), 1,010 CMD subjects had diabetes, 1,919 had hypertension, and 738 had coronary heart disease (CHD). Follow-up until Dec 31, 2019 found that in CMD-affected individuals, longevity-associated alleles of FOXO3 were associated with significantly longer lifespan: haplotype hazard ratio 0.81 (95% CI 0.72-0.91; diabetes 0.77, hypertension 0.82, CHD 0.83). Overall, men with a CMD had higher mortality than men without a CMD (P=6x10-7). However, those men with a CMD who had the FOXO3 longevity genotype had similar survival as men without a CMD. In men without a CMD there was no association of longevity-associated alleles of FOXO3 with lifespan. Our study provides novel insights into the basis for the long-established role of FOXO3 as a longevity gene. We suggest that the FOXO3 longevity genotype increases lifespan only in at-risk individuals by protection against cardiometabolic stress.

To investigate interindividual differences in cognitive terminal decline and identify determinants including functional, health, and genetic risk and protective factors, data from the Honolulu Heart Program/Honolulu-Asia Aging Study, a prospective cohort study of Japanese American men, were analyzed. The sample was recruited in 1965–1968 (ages 45–68 years). Longitudinal performance of cognitive abilities and mortality status were assessed from Exam 4 (1991–1993) through June 2014. Latent class analysis revealed 2 groups: maintainers retained relatively high levels of cognitive functioning until death and decliners demonstrated significant cognitive waning several years prior to death. Maintainers were more likely to have greater education, diagnosed coronary heart disease, and presence of the apolipoprotein E (APOE) ε2 allele and FOXO3 G allele (SNP rs2802292). Decliners were more likely to be older and have prior stroke, Parkinson’s disease, dementia, and greater depressive symptoms at Exam 4, and the APOE ε4 allele. Findings support terminal decline using distance to death as the basis for modeling change. Significant differences were observed between maintainers and decliners 15 years prior to death, a finding much earlier compared to the majority of previous investigations.

Three-dimensional chromatin architecture and gene-gene interactions impact gene expression. We assembled this information, in silico, for the human renin gene (REN). We searched for chromatin contacts and boundaries and the locations of super-enhancers that are involved in cell specific differentiation. The REN promoter was connected via RNA polymerase II binding to promoters of 12 neighboring genes on chromosome 1q32.1 over a distance of 762,497 bp. This constitutes a regulatory archipelago. The genes formed 3 topologically associated domains (TADs), as follows: TAD1: ZC3H11A, SNRPE, LINC00303; SOX13; TAD2: ETNK2, REN, KISS1, GOLT1A; TAD3: PLEKHA6, LINC00628, PPP1R15B, PIK3C2B, MDM4. REN in TAD2, was isolated from its neighboring genes in TAD1 and TAD3 by CTCF-binding sites that serve as insulators. TAD1 and TAD3 genes SOX13 and LINC00628 overlapped super-enhancers, known to reside near nodes regulating cell identity, and were co-expressed in various tissues, suggesting co-regulation. REN was also connected with 62 distant genes genome-wide, including the angiotensin II type 1 receptor gene. The findings lead us to invoke the following novel hypothesis. While the REN promoter is isolated from neighboring super-enhancers in most cells by insulators, these insulators break down with cell age to permit the inappropriate expression of REN in non-kidney cells by using the neighboring super-enhancers, resulting in expression in a wider spectrum of tissues, contributing to aging-related immune system dysregulation, cardiovascular diseases and cancers. Research is needed to confirm this hypothesis experimentally.

The rs2802292, rs2764264 and rs13217795 variants of FOXO3 have been associated with extreme longevity in multiple human populations, but the mechanisms underpinning this remain unclear. We aimed to characterise potential effects of longevity-associated variation on the expression and mRNA processing of the FOXO3 gene. We performed a comprehensive assessment of FOXO3 isoform usage across a wide variety of human tissues and carried out a bioinformatic analysis of the potential for longevity-associated variants to disrupt regulatory regions involved in isoform choice. We then related the expression of full length and 5′ truncated FOXO3 isoforms to rs13217795 genotype in peripheral blood and skeletal muscle from individuals of different rs13217795 genotypes. FOXO3 isoforms displayed considerable tissue specificity. We determined that rs13231195 and its tightly aligned proxy variant rs9400239 may lie in regulatory regions involved in isoform choice. The longevity allele at rs13217795 was associated with increased levels of full length FOXO3 isoforms in peripheral blood and a decrease in truncated FOXO3 isoforms in skeletal muscle RNA. We suggest that the longevity effect of FOXO3 SNPs may in part derive from a shift in isoform usage in skeletal muscle away from the production of 5′ truncated FOXO3 isoforms lacking a complete forkhead DNA binding domain, which may have compromised functionality.

Chromosomal translocations occur in 10 to 15% of men with azoospermia. Thirty distinct X-autosomal balanced reciprocal translocations have been reported in the literature thus far. We present a novel case of azoospermia with a karyotype of 46,Y,t(X:16)(p22.1:p11.2). A 26-year-old, healthy, active duty male Solider presented with his dependent female partner for primary infertility. Female anatomical and endocrine evaluations were normal. Initial male evaluation revealed azoospermia on multiple semen analyses. Further evaluation with a detailed physical exam and laboratory tests were normal except for an abnormal karyotype with a reciprocal translocation at chromosomes X and 16. An open testicular biopsy demonstrated 75% late spermatid maturation arrest confirming reproductive potential although significantly reduced. Men who present with azoospermia should undergo a full endocrine and genetic evaluation with a thorough physical evaluation by an urologist. They can have limited but successful reproductive outcomes if spermatozoa can be isolated during testicular biopsy. Given the high risk of producing genetically unbalanced embryos, genetic counseling and preimplantation genetic testing is essential before pursuing assisted reproductive technology. This case is the first X-autosomal balanced reciprocal translocations involving chromosome 16 and highlights the importance of the X chromosome during spermatogenesis.

The original version of this chapter was inadvertently published with only one affiliation for Philip M. C. Davy. Additional affiliation has now been included in chapter metadata and front matter.

Summary FOXO3 has been implicated in longevity in multiple populations. By DNA sequencing in long‐lived individuals, we identified all single nucleotide polymorphisms (SNPs) in FOXO3 and showed 41 were associated with longevity. Thirteen of these had predicted alterations in transcription factor binding sites. Those SNPs appeared to be in physical contact, via RNA polymerase II binding chromatin looping, with sites in the FOXO3 promoter, and likely function together as a cis‐regulatory unit. The SNPs exhibited a high degree of LD in the Asian population, in which they define a specific longevity haplotype that is relatively common. The haplotype was less frequent in whites and virtually nonexistent in Africans. We identified distant contact points between FOXO3 and 46 neighboring genes, through long‐range physical contacts via CCCTC‐binding factor zinc finger protein (CTCF) binding sites, over a 7.3 Mb distance on chromosome 6q21. When activated by cellular stress, we visualized movement of FOXO3 toward neighboring genes. FOXO3 resides at the center of this early‐replicating and highly conserved syntenic region of chromosome 6. Thus, in addition to its role as a transcription factor regulating gene expression genomewide, FOXO3 may function at the genomic level to help regulate neighboring genes by virtue of its central location in chromatin conformation via topologically associated domains. We believe that the FOXO3 ‘interactome’ on chromosome 6 is a chromatin domain that defines an aging hub. A more thorough understanding of the functions of these neighboring genes may help elucidate the mechanisms through which FOXO3 variants promote longevity and healthy aging.

Summary The G allele of the FOXO3 single nucleotide polymorphism (SNP) rs2802292 exhibits a consistently replicated genetic association with longevity in multiple populations worldwide. The aims of this study were to quantify the mortality risk for the longevity-associated genotype and to discover the particular cause(s) of death associated with this allele in older Americans of diverse ancestry. It involved a 17-year prospective cohort study of 3584 older American men of Japanese ancestry from the Honolulu Heart Program cohort, followed by a 17-year prospective replication study of 1595 white and 1056 black elderly individuals from the Health Aging and Body Composition cohort. The relation between FOXO3 genotype and cause-specific mortality was ascertained for major causes of death including coronary heart disease (CHD), cancer, and stroke. Age-adjusted and multivariable Cox proportional hazards models were used to compute hazard ratios (HRs) for all-cause and cause-specific mortality. We found G allele carriers had a combined (Japanese, white, and black populations) risk reduction of 10% for total (all-cause) mortality (HR = 0.90; 95% CI, 0.84–0.95; P = 0.001). This effect size was consistent across populations and mostly contributed by 26% lower risk for CHD death (HR = 0.74; 95% CI, 0.64–0.86; P = 0.00004). No other causes of death made a significant contribution to the survival advantage for G allele carriers. In conclusion, at older age, there is a large risk reduction in mortality for G allele carriers, mostly due to lower CHD mortality. The findings support further research on FOXO3 and FoxO3 protein as potential targets for therapeutic intervention in aging-related diseases, particularly cardiovascular disease.

Background: Although numerous studies have been published on prognostic factors after a first acute myocardial infarction (AMI) among middle-aged men, little is known about the prognostic factors and genetic determinants for post-AMI elderly patients. Methods: The Kuakini Honolulu Heart Program (KHHP) is a prospective population-based study of cardiovascular disease (CVD) (defined as coronary heart disease (CHD) and stroke) among men of Japanese ancestry living in Hawaii. We identified 141 first AMI patients from participants of the KHHP exam 4 (1991-93) by ECG and/or cardiac enzymes (onset age: 73-96 years). Men who survived more than one year after AMI were followed for mortality from onset of AMI to December 2018 (all were deceased by 28 years of follow-up). All had FOXO3 genotype information available. Quantile regression was used to compare the median survival times of FOXO3-G allele carriers (longevity genotype) with FOXO3-TT homozygotes (common genotype). Results: Adjusting for age at AMI onset, baseline body mass index (BMI) and glucose levels (at exam 4), median survival times differed between FOXO3-G allele carriers and FOXO3-TT homozygotes by 2.1 years (95% CI=0.24-3.95, p=0.027). Adding other known CHD risk factors (i.e. hypertension, etc.) to the model reduced the difference to 2.03 years (95% CI=0.17-3.89, p=0.033). Furthermore, chi-square testing showed the mortality rate from CVD among FOXO3-G allele carriers was significantly lower than that among the common FOXO3-TT genotype. Conclusions: The data suggest that the FOXO3 longevity genotype reduces the risk of dying from CVD and extends survival time of elderly patients with acute myocardial infarction.

3,734 Japanese-American male oldest-old (aged 85+ years), from the Kuakini Honolulu Asia Aging Study, were assessed for prevalent cognitive impairment (CI). CI was defined as scoring

Next-generation DNA sequencing has ushered in a new era of genotype-phenotype comparisons that have the potential to elucidate the genetic nature of complex traits. Since such methods rely on short sequence reads and since the human genome is composed largely of repetitive DNA elements larger than these read lengths many results cannot be mapped and are discarded, thus eliminating a large portion of the genome from analysis. Discerning associations in complex traits, such as longevity, will require either longer read lengths or methods to address these sequence complexities. Whole genome analysis, such as Genome Wide Association Studies (GWAS), also suffers from the repetitive nature of the human genome, as there exist many gaps in the availability of useable genetic markers, often in interesting regulatory regions. Methods are described here whereby some of these problems have been addressed by targeted DNA sequencing, full exploitation of available public databases, and a careful evaluation of genomic features where we use the FOXO3 gene as an example to identify functional variations and how they may relate to longevity.

Here we summarize the latest data on genetic and epigenetic contributions to human aging and longevity. Whereas environmental and lifestyle factors are important at younger ages, the contribution of genetics appears more important in reaching extreme old age. Genome-wide studies have implicated ~57 gene loci in lifespan. Epigenomic changes during aging profoundly affect cellular function and stress resistance. Dysregulation of transcriptional and chromatin networks is likely a crucial component of aging. Large-scale bioinformatic analyses have revealed involvement of numerous interaction networks. As the young well-differentiated cell replicates into eventual senescence there is drift in the highly regulated chromatin marks towards an entropic middle-ground between repressed and active, such that genes that were previously inactive “leak”. There is a breakdown in chromatin connectivity such that topologically associated domains and their insulators weaken, and well-defined blocks of constitutive heterochromatin give way to generalized, senescence-associated heterochromatin, foci. Together, these phenomena contribute to aging.

Aging is a complex, multifactorial process with significant plasticity. While several biological pathways appear to influence aging, few genes have been identified that are both evolutionarily conserved and have a strong impact on aging and age-related phenotypes. The FoxO3 gene (FOXO3), and its homologs in model organisms, appears especially important, forming a key gene in the insulin/insulin-like growth factor-signaling pathway, and influencing life span across diverse species. We highlight some of the key findings that are associated with FoxO3 protein, its gene and homologs in relation to lifespan in different species, and the insights these findings might provide about the molecular, cellular, and physiological processes that modulate aging and longevity in humans.

The minor alleles of 3 FOXO3 single nucleotide polymorphisms (SNPs)- rs2802292 , rs2253310 , and rs2802288 -are associated with human longevity. The aim of the present study was to test these SNPs for association with blood pressure (BP) and essential hypertension (EHT).In a primary study involving Americans of Japanese ancestry drawn from the Family Blood Pressure Program II we genotyped 411 female and 432 male subjects aged 40-79 years and tested for statistical association by contingency table analysis and generalized linear models that included logistic regression adjusting for sibling correlation in the data set. Replication of rs2802292 with EHT was attempted in Japanese SONIC study subjects and of each SNP in a meta-analysis of genome-wide association studies of BP in individuals of European ancestry.In Americans of Japanese ancestry, women homozygous for the longevity-associated (minor) allele of each FOXO3 SNP had 6mm Hg lower systolic BP and 3mm Hg lower diastolic BP compared with major allele homozygotes (Bonferroni corrected P0.05 and0.05, respectively). Frequencies of minor allele homozygotes were 3.3-3.9% in women with EHT compared with 9.5-9.6% in normotensive women ( P = 0.03-0.04; haplotype analysis P = 0.0002). No association with BP or EHT was evident in males. An association with EHT was seen for the minor allele of rs2802292 in the Japanese SONIC cohort ( P = 0.03), while in European subjects the minor allele of each SNP was associated with higher systolic and diastolic BP.Longevity-associated FOXO3 variants may be associated with lower BP and EHT in Japanese women.

Background: The gene FOXO3, encoding the transcription factor forkhead box O-3 (FoxO3), is one of only two for which genetic polymorphisms have exhibited consistent associations with longevity in diverse human populations. Objective: Here, we review the multitude of actions of FoxO3 that are relevant to health, and thus healthy ageing and longevity. Methods: The study involved a literature search for articles retrieved from PubMed using FoxO3 as keyword. Results: We review the molecular genetics of FOXO3 in longevity, then current knowledge of FoxO3 function relevant to ageing and lifespan. We describe how FoxOs are involved in energy metabolism, oxidative stress, proteostasis, apoptosis, cell cycle regulation, metabolic processes, immunity, inflammation and stem cell maintenance. The single FoxO in Hydra confers immortality to this fresh water polyp, but as more complex organisms evolved, this role has been usurped by the need for FoxO to control a broader range of specialized pathways across a wide spectrum of tissues assisted by the advent of as many as 4 FoxO subtypes in mammals. The major themes of FoxO3 are similar, but not identical, to other FoxOs and include regulation of cellular homeostasis, particularly of stem cells, and of inflammation, which is a common theme of age-related diseases. Other functions concern metabolism, cell cycle arrest, apoptosis, destruction of potentially damaging reactive oxygen species and proteostasis. Conclusions: The mechanism by which longevity-associated alleles of FOXO3 reduce age-related mortality is currently of great clinical interest. The prospect of optimizing FoxO3 activity in humans to increase lifespan and reduce age-related diseases represents an exciting avenue of clinical investigation. Research strategies directed at developing therapeutic agents that target FoxO3, its gene and proteins in the pathway(s) FoxO3 regulates should be encouraged and supported.

The transcription factor FoxO3 regulates multiple genes involved in cell resilience. We have previously implicated variation in non-coding DNA of the FoxO3 gene ( FOXO3 ) with lower blood pressure, reduced inflammation, less hypertension, reduced coronary heart disease mortality, and longevity. The aim of the present study was to determine transcriptional, genetic and genomic mechanisms involving FOXO3 . By DNA sequencing of chromosome 6q21 in lymphoblastoid cell lines of 95 men who had survived to ≥ 95 years of age we identified 110 FOXO3 single nucleotide polymorphisms (SNPs). Thirteen SNPs were at binding sites for 18 transcription factors. Those SNPs appeared to be in physical contact, via RNA polymerase II binding chromatin looping, with sites in the FOXO3 promoter, and likely function together as a cis -regulatory unit. At the chromosome level, FOXO3 was located at the center of a 7.3 Mb 46-gene chromatin domain flanked by gene deserts. We identified distant contact points between FOXO3 and these 46 neighboring genes, through long-range physical contacts via CCCTC-binding factor zinc finger protein (CTCF) binding sites. The genes in this “archipelago” of neighbourhood genes mediate a similar repertoire of functions as FoxO3, including stress resistance, nutrient sensing, cell proliferation, autophagy, apoptosis and stem cell maintenance. The 7.3 Mb gene domain was highly conserved across species, indicating evolutionary importance. We believe that FOXO3 serves as the hub for an “interactome” involved in healthy aging, including cardiovascular disease reduction, in those with favorable FOXO3 genotypes. In support, we found that cellular stress (H 2 O 2 ) could stimulate FOXO3 expression in 20 lymphoblastoid cell lines, being 3-fold stronger for those with a favorable FOXO3 genotype. In FISH experiments, stress-induced activation of FOXO3 caused it to move towards its neighboring genes as suggested by our genomic data. In conclusion, we have shown, for the first time, that FOXO3 is at the central hub of a gene network on chromosome 6 involved in cell protection and healthy aging. The concept of “gene factories” may apply more broadly to genome and genetic mechanisms involved in cardiovascular disease etiology.

Longevity is a polygenic trait in which genetic predisposition is particularly important. We hypothesized that among genes differentially expressed in response to caloric restriction, several may be candidate longevity genes. We tested 459 single-nucleotide polymorphisms (SNPs) in 47 genes differentially expressed in calorically restricted mice and 12 other genes for association with longevity. Subjects were American men of Japanese ancestry, 440 aged ≥95 years and 374 with an average life span. Based on a dominant model of inheritance, an association with longevity at the p < .05 level was seen for SNPs in 13 of the genes. Testing by all possible models increased the number of genes to 18. After correction for multiple testing, four genes retained significance, namely, MAP3K5 (p = .00004), SIRT7 (p = .00004), SIRT5 (p = .0007), and PIK3R1 (p = .01). In a dominant model, association with longevity was seen for multiple adjacent SNPs within two of these genes (MAP3K5 and PIK3R1), as well as in FLT1, consistent with linkage disequilibrium with a causative variant in the vicinity of each respective SNP set. MAP3K5 and FLT1 haplotypes were associated with longevity. In conclusion, the present study implicates variation in MAP3K5, FLT1, PIK3R1, SIRT7, and SIRT5 in human longevity.

FOXO3 is one of the most prominent genes demonstrating a consistently reproducible genetic association with human longevity. The mechanisms by which these individual gene variants confer greater organismal lifespan are not well understood. We assessed the effect of longevity-associated FOXO3 alleles on age-related leukocyte telomere dynamics in a cross-sectional study comprised of samples from 121 healthy Okinawan-Japanese donors aged 21-95 years. We found that telomere length for carriers of the longevity associated allele of FOXO3 single nucleotide polymorphism rs2802292 displayed no significant correlation with age, an effect that was most pronounced in older (>50 years of age) participants. This is the first validated longevity gene variant identified to date showing an association with negligible loss of telomere length with age in humans in a cross-sectional study. Reduced telomere attrition may be a key mechanism for the longevity-promoting effect of the FOXO3 genotype studied.

Objectives: To identify potentially modifiable late-life biological, lifestyle, and sociodemographic factors associated with overall and healthy survival to age 85. Design: Prospective longitudinal cohort study with 21 years of follow-up (1991�2012). Setting: Hawaii Lifespan Study. Participants: American men of Japanese ancestry (mean age 75.7, range 71�82) without baseline major clinical morbidity and functional impairments (N = 1,292). Measurements: Overall survival and healthy survival (free from six major chronic diseases and without physical or cognitive impairment) to age 85. Factors were measured at late-life baseline examinations (1991�1993). Results: Of 1,292 participants, 1,000 (77%) survived to 85 (34% healthy) and 309 (24%) to 95 (

Age is the most powerful independent risk factor for major causes of death in developed countries. The rate of aging is influenced by environmental and genetic factors. Identification of the molecular mechanisms responsible for this complex polygenic phenotype represents a major current challenge. Because the genetic component becomes paramount for the attainment of extreme old age (1–3) and caloric restriction is the most effective means of extending life span in diverse species (4), studies of polymorphisms in genes belonging to intracellular pathways that are modulated by caloric restriction have been conducted in nonagenarian and centenarian populations (4,5). These pathways affect insulin/insulin-like growth factor-1 signaling and growth responses. The hyperfunction theory of aging postulates that in later life, the once-beneficial processes contributing to growth become deleterious by causing hypertrophic and hyperplastic pathologies (6). The mechanistic (formerly “mammalian”) target of rapamycin (mTOR) is positioned at a central hub of nutrient-sensing pathways (7,8) and is pivotal to life span extension in response to caloric restriction (9,10). Despite good evidence showing that insulin/insulin-like growth factor-1 and mTOR pathways are critical for life span regulation of model organisms (6,11,12), to date, attainment of extreme old age in humans has been consistently replicated only for variation in one gene in these pathways, the forkhead box O transcription factor 3 gene (FOXO3) (5,6). mTOR is an evolutionarily conserved serine/threonine phosphoinositide 3-kinase-related kinase with crucial roles in cell growth and metabolism in response to nutrients, growth factors, cellular energy, and stress (6,8,13,14). Rapamycin forms a stoichiometric complex with mTOR to inhibit its kinase activity (15,16) and can thus extend mean and maximum life span of middle-aged male and female mice, with no adverse health consequences, only benefits (17,18). mTOR is a component of two structurally and functionally distinct multiprotein complexes—TOR complex 1 (TORC1) and TOR complex 2 (TORC2). In mammals, two accessory proteins—regulatory-associated protein of mTOR (raptor or RPTOR) and rapamycin-insensitive companion of mTOR (rictor or RICTOR)—distinguish TORC1 from TORC2, respectively (19,20). mTORC1 exerts its effects on temporal control of cell growth by regulating several cellular processes, including translation, transcription, ribosome biogenesis, nutrient transport, and autophagy (14). The two TOR complexes constitute an evolutionarily conserved ancestral signaling network that responds to energy levels and sources of energy in order to control the fundamental process of cell growth and maintenance. In addition to its key role in aging, mTOR has been implicated in cancer, cardiovascular disease, obesity, and diabetes (21). Rapamycin inhibits TORC1 by forming a complex with FK506-binding protein (FKBP12), which then binds to and inhibits TORC1 in its complex with raptor (16,22). In contrast, rapamycin does not bind to TORC2. Binding of raptor to eukaryotic initiation factor 4E-binding protein-1 stimulates ribosomal protein S6 kinase (23) and ribosomal protein S6 kinase, 90kDa, polypeptide 1 (RPS6KA1). RPS6KA1 is a downstream effector of nutrient-responsive mTOR signaling. It regulates multiple transcription factors, increases cell growth—in part by increasing mTOR signaling—and links nutrient availability to aging in organisms as diverse as yeast and mice (24). Rapamycin reduces mTOR-mediated phosphorylation of RPS6KA1 (16), so increasing life span and resistance to age-related pathologies, as well as producing gene expression patterns similar to those seen in caloric restriction or pharmacological activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), a conserved regulator of the metabolic response to caloric restriction (25). Here we report the results of a genetic association study of tagging single-nucleotide polymorphisms (tagSNPs) that provided virtually complete coverage of key mTOR complex genes MTOR, RPTOR, and RICTOR, as well as RPS6KA1, in long-lived Americans of Japanese ancestry. The study tested for association of these genetic variants with living to more than 95 years of age and with multiple aging-related parameters in these subjects.

Evidence from model organisms suggests that the insulin/IGF-1 signaling pathway has an important, evolutionarily conserved influence over rate of aging and thus longevity. In humans, the FOXO3 gene is the only widely replicated insulin/IGF-1 signaling pathway gene associated with longevity across multiple populations. Therefore, we conducted a nested case-control study of other insulin/IGF-1 signaling genes and longevity, utilizing a large, homogeneous, long-lived population of American men of Japanese ancestry, well characterized for aging phenotypes. Genotyping was performed of single nucleotide polymorphisms, tagging most of the genetic variation across several genes in the insulin/IGF-1 signaling pathway or related gene networks that may be influenced by FOXO3, namely, ATF4, CBL, CDKN2, EXO1, and JUN. Two initial, marginal associations with longevity did not remain significant after correction for multiple comparisons, nor were they correlated with aging-related phenotypes.

Background We recently reported that protection against coronary artery disease (CAD) mortality is the major contributor to longer life associated with FOXO3 genotype. The present study examined this relation in more detail. Methods We performed a 15-year observational study of 3,584 older American men of Japanese ancestry from the Kuakini Honolulu Heart Program cohort and 1,595 White and 1,067 Black elderly individuals from the Health Aging and Body Composition study. Results Multivariate Cox regression models demonstrated that carriage of the longevity-associated G allele of FOXO3 single nucleotide polymorphisms rs2802292 was a protective factor against CAD mortality in all three populations. In Japanese and Whites, but not in Blacks, the protective effect of the G allele was little changed in models adjusted for other major risk factors. Population-attributable risk (PAR) models found that the nonprotective TT genotype contributed 15%, 9%, and 3% to CAD mortality risk in Japanese, White, and Black Americans, respectively, and was one of the top three contributing factors to CAD mortality. In Japanese, this effect size was comparable with hypertension (15%), but in Whites and Blacks PAR for hypertension was higher (29% and 26%, respectively). G-allele carriers had lower plasma TNF-α than noncarriers, suggesting inflammation as a potential mediating factor for CAD mortality risk. Conclusion FOXO3 genotype is an important risk factor for CAD mortality in older populations. More research is needed to identify potential mechanisms and targets for intervention.

There are several methods for measuring healthy or “successful” aging utilizing different measures of comorbidity. Each can lead to different conclusions about how “successfully” one is aging. To bring more clarity, we compared 3,741 American men of Japanese ancestry (mean age 77.8 ± 4.7, range 71–93 years) from the 1991–94 exam of the Kuakini Honolulu Asia Aging Study in a cross-sectional analysis, utilizing the Rowe and Kahn criteria versus a modified Charlson Index. With Rowe and Kahn criteria, 50.4%, 34.3% and 7.3% of men in their 70s, 80s and 90s, were successful agers, respectively (p

Prohormone convertase subtilisin/kexin type 1 (PCSK1) genetic polymorphisms have recently been associated with obesity in European populations. This study aimed to examine whether common PCSK1 genetic variation is associated with obesity and related metabolic phenotypes in the Chinese population. We genotyped nine common tag single-nucleotide polymorphisms (tagSNP) of the PCSK1 gene in 1,094 subjects of Chinese origin from the Stanford Asia-Pacific Program for Hypertension and Insulin Resistance (SAPPHIRe) family study. One SNP in the PCSK1 gene (rs155971) were nominally associated with risk of obesity in the SAPPHIRe cohort (P = 0.01). A common protective haplotype was associated with reduced risk of obesity (23.79% vs. 32.89%, P = 0.01) and smaller waist circumference (81.71 +/- 10.22 vs. 84.75 +/- 10.48 cm, P = 0.02). Another common haplotype was significantly associated with increased risk of obesity (37.07% vs. 23.84%, P = 0.005). The global P value for haplotype association with obesity was 0.02. We also identified a suggestive association of another PCSK1 SNP (rs3811951) with fasting glucose, fasting insulin, homeostasis model assessment of insulin resistance (HOMA(IR)), triglycerides, and high-density lipoprotein cholesterol (P = 0.05, 0.003, 0.001, 0.04, and 0.04, respectively). These data indicate common PCSK1 genetic variants are associated with obesity in the Chinese population.

Objectives. To investigate the reliability and correlations with age of the balance components of the EPESE, NHANES, and the Good Balance Platform System (GBPS) in a normal population of adults.Design. Cross-sectional.Setting. Urban Medical Center in the Pacific.Participants. A random sample of 203 healthy offspring of Honolulu Heart Program participants, ages 38–71.Measurements. Subjects were examined twice at visits one week apart using the balance components of the EPESE, NHANES, and the good balance system tests.Results. The EPESE and NHANES batteries of tests were not sufficiently challenging to allow successful discrimination among subjects in good health, even older subjects. The GBPS allowed objective quantitative measurements, but the test-retest correlations generally were not high. The GBPS variables correlated with age only when subjects stood on a foam pad; they also were correlated with anthropometric variables.Conclusion. Both EPESE and NHANES balance tests were too easy for healthy subjects. The GBPS had generally low reliability coefficients except for the most difficult testing condition (foam pad, eyes closed). Both height and body fat were associated with GBPS scores, necessitating adjusting for these variables if using balance as a predictor of future health.

Background High-density lipoprotein cholesterol (HDL-C) and cholesteryl ester transfer protein (CETP) gene deficiency mutations that increase HDL-C levels have been associated with exceptional longevity. However, a recent clinical trial of a promising CETP inhibitor that markedly increases HDL-C was terminated due to increased mortality. In light of this controversy, we examined the relationship among HDL-C, CETP mutations, and longevity phenotypes in the long-lived Japanese-American men of the Honolulu Heart Program (HHP). Methods Japanese-American men (n = 3562) were followed for up to 8 years, from average age 78 to average age 84 (maximum age 99), or until death. Total mortality, cause-specific mortality, and healthy survival were evaluated for associations with HDL-C level and CETP genetic variants common in the Japanese population (CD442G and Int 14A). Results HDL-C was negatively associated with cardiovascular disease (CVD) mortality (p =.002) but not related to non-CVD (p =.147) or total (p =.547) mortality after adjustment for common risk factors. There was a trend for lower mortality for the men with the Int 14A variant. These men also had higher HDL-C levels (p =.047) and were significantly more likely to be healthy survivors (absence of six major age-related diseases and high physical/cognitive function) beyond the age of 90 years (p =.005). Conclusions Low HDL-C level is a risk factor for CVD mortality in elderly Japanese-American men. High HDL-C and the Int 14A variant of the CETP gene may increase odds for healthy aging.

Aims Although the function of resistin in human biology is unclear, some evidence suggests resistin gene variants influence insulin resistance, and insulin resistance-related hypertension. We searched for associations between common resistin gene variants and factors related to insulin resistance in Asian individuals with high or low blood pressure (BP). Methods Non-diabetic Chinese or Japanese sibling pairs were included if one had extreme hypertension and the other was either hypertensive or hypotensive. Four common, non-coding single nucleotide polymorphisms (SNPs) were identified by sequencing the resistin gene in 24 hypertensive probands. Generalized estimating equations (GEEs)-based regressions were then performed to test for SNP associations using the entire study population (n = 1556). Results Of 72 tests, only one was significant at the 0.05 level; 3.5 significant tests were expected by chance alone. High variability in insulin and triglyceride levels created wide confidence intervals, thus the negative results are not conclusive for these phenotypes. However, the large sample size resulted in narrow confidence intervals for BMI, fasting and 120 min post-load glucose, and high and low density lipoprotein cholesterol (LDL-C). Conclusion Several factors associated with insulin resistance are not likely influenced by the resistin gene in non-diabetic Asian individuals with high and low blood pressure.

The G allele of the FOXO3 SNP rs2802292 has a strong, consistently replicated protective association with longevity. We investigated the effect of FOXO3 genotype on coronary heart disease (CHD) mortality - a major cause of mortality worldwide. We genotyped DNA collected in 1991-93, at Exam 4 of the Honolulu Heart Program (HHP), from 3,584 American males of Japanese ancestry aged 71-93 years. Study participants were followed for up to 9 years for CHD mortality and for up to 11 years in a replication cohort - the Health Aging and Body Composition (Health ABC) study. Health ABC is a prospective observational study of 3,075 well functioning, community-dwelling, male and female, white and black Americans, aged 70-79 years at baseline. Cox proportional hazards models demonstrated that Americans of Japanese, white and black ethnicity who were G allele carriers had a combined risk reduction of 31% for CHD mortality (HR = 0.69; 95% CI, 0.58-0.82; P =0.00002). Reduction in risk of death from CHD was 38% in Japanese, 24% in whites and 29% in blacks. This finding remained virtually unchanged when included in a multivariable model of common CHD risk factors. We subsequently assessed the contribution of FOXO3 genotype, relative to other common risk factors, to CHD mortality in these populations by estimating population attributable risk (PAR). In the Americans of Japanese ancestry, PAR for the TT (non-protective) genotype was high, at 24%, equivalent to hypertension (24%), and greater than diabetes (21%), hypercholesterolemia (12%) and ever smoking (6%). PAR for FOXO3 genotype was lower at 9% and 3% in white and black Americans, respectively, principally due to lower prevalence of the TT (risk) genotype. We subsequently measured C-reactive protein (CRP) in a population subsample, and found lower blood CRP levels in G allele carriers ( P G allele of the FOXO3 SNP rs2802292 has a major protective effect on CHD mortality, possibly related to lower blood levels of inflammation. The findings support further research on potential mechanisms and point to FoxO3 as a potential target therapeutic target.

Background Lipid levels are recognized as major risk factors for coronary heart disease (CHD). Discovery of major loci underlying quantitative lipid traits could help to elucidate the genetics of CHD. Methods We performed a genome-wide search for quantitative trait loci linked to lipid phenotypes in 1538 Chinese subjects (509 families) and 625 Japanese subjects (204 families) not taking lipid-lowering medications from the Stanford-Asian Pacific Program in Hypertension and Insulin Resistance (SAPPHIRe) study. The multipoint variance-components method was used to test for linkage between marker loci and each trait by maximum likelihood methods adjusted for effects of age, age2, gender, body mass index (BMI), smoking, alcohol drinking, physical activity, and field center. Results The highest logarithm of odds (LOD) score detected was 3.22 for logarithmically transformed HDL-cholesterol on chromosome 12 at 113 cM in Chinese subjects. This score overlaps the positive findings for HDL reported in Mexican Americans (chromosome 12 at 96 cM). Although no strong evidence for linkage was found in Japanese subjects, some modest peaks (LOD score ≧1.5) were found in several regions that have been reported in other published genome scans. For example, the Japanese SAPPHIRe peak for HDL (chromosome 1 at 167 cM; LOD = 1.54) was very close to the quantitative trait loci (QTL) for HDL reported in the scan of white American HyperGEN (chromosome 1 at 159.9 cM). Conclusions Genome-wide scan for genes influencing lipid phenotypes was conducted and we found significant linkage of HDL to a locus on chromosome 12 in Chinese subjects and no linkage signal exceeding 1.68 was found in the Japanese subjects.

OBJECTIVES: To improve and broaden the applicability of performance-based measures of function for use in clinical and research settings. DESIGN: Cross-sectional repeated-measures study. SETTING: Research clinic. PARTICIPANTS: Population-based sample of Japanese Americans without significant functional impairments aged 35 to 55 and 56 to 71 (N=203). MEASUREMENTS: Performance-based measures of physical function, including range of standard tests, newer automated measures of balance and strength and data on cognitive function, lifestyle, medical history, and physical activity. RESULTS: Of the nonplatform balance measures, only the one-leg stand was reliable (reliability coefficient (rc) =0.69) and able to discriminate between functional levels. Combining the Fourth National Health and Nutrition Examination Survey protocol of folded arm position while standing on a foam pad with the more-sophisticated balance platform test had the highest reliability and discrimination. With the strength chair, high rcs (0.88–0.96) were found for upper and lower extremity tests. Timed chair stands, a test of lower extremity and central strength were reliable. The 6-minute walk had a high rc (0.90). CONCLUSION: Many performance tests used today are not reliable. Only a few discriminate between the most highly functioning individuals and individuals with good function. Thus, a new recommended battery includes unassisted single-leg stand, balance platform “foam pad, eyes closed,” elbow flexion and knee extension strength (strength chair), grip strength, timed chair stands, and the 6-minute walk. These simple performance-based tests have good reliability and discrimination across the range of function and can be used in most clinical and research settings to quickly assess global functional level.

Common polymorphisms in genes that affect estrogen levels may be associated with breast cancer risk. We investigated the associations between breast cancer and sequence variants in several genes in the estradiol/estrone metabolism pathway (CYP1A1*2A, CYP1A2*1F, CYP1B1 Leu432Val, CYP3A4*1B, COMT Val158Met, SULT1A1Arg213His) as well as the Arg554Lys variant in AHR (a transcription factor for CYP1A1, CYP1A2, and CYP1B1) in a case-control study of 1,339 breast cancer cases and 1,370 controls nested in the Multiethnic Cohort Study. The Multiethnic Cohort Study is a large prospective study of men and predominantly postmenopausal women of Japanese, White, African American, Latino, and Native Hawaiian ancestry, residing in Hawaii and Los Angeles. We found no association between breast cancer and these polymorphisms, except for CYP1A2*1F which was inversely associated with risk. The odds ratio (95% confidence interval) for the AA, AC, and CC genotype was 1.0, 0.9 (0.7-1.0), and 0.7 (0.5-1.0), respectively (P for gene dosage effect = 0.03). This association seemed somewhat stronger for estrogen receptor (ER)/progesterone receptor (PR)–negative tumors than for ER/PR-positive tumors, and no statistically significant interaction with estrogen-related risk factors was detected. The findings provide no evidence for a role of COMT Val58Met, CYP1A1*2A, CYP3A4*1B, CYP1B1 Leu432Val, SULT1A1 Arg213His, and AHR Arg554Lys in breast cancer etiology. They also provide support for an inverse association between CYP1A2*1F and breast cancer, which is consistent with the observation of lower circulating estrogen levels in premenopausal women with the CC genotype in a previous study.